The interests of this section are in the area of protein structure as it relates to function, with a focus on the interactions of components involved in cell differentiation survival and maintenance. Our research at NEI has applied these interests to systems in the retina.
Our section has been studying pigment epithelium-derived factor (PEDF), a protein that acts in neuronal differentiation and survival in cells derived from the retina and CNS. PEDF inhibits angiogenesis and its expression is down- regulated over the replicative lifespan of mammals. This interesting factor is secreted by retinal pigment epithelial cells into the interphotoreceptor matrix, where it acts on photoreceptor cells. Its importance in the development, maintenance, and function of the retina and CNS is evident in animal models for inherited and light-induced retinal degeneration, as well as for degeneration of spinal cord motor neurons.
Much of our progress in understanding PEDF has relied on our development of overexpression systems that yielded recombinant proteins as functionally active neurotrophic factors identical to the native protein and ideal for biochemical, biophysical, and biological studies. The cDNA sequence for PEDF predicts a unique protein with strong homology to members of the serine protease inhibitor (serpin) superfamily. For this reason, we first studied PEDF as a serpin. We established that PEDF belongs with the subgroup of noninhibitory serpins and that it has characteristics of a substrate rather than an inhibitor of serine proteases. Moreover, a region from its amino-terminus confers the neurotrophic activity to the PEDF polypeptide, not requiring the serpin reactive loop (the structural determinant for serpin inhibition). We concluded that PEDF’s neurotrophic activity must be mediated by a mechanism independent of serine protease inhibition, and we proposed that during evolution this serpin might have lost its inhibitory activity and gained neurotrophic function. These findings provided an example of the separation of inhibitory and other activities in a serpin.
Our investigations were next directed toward the hypothesis that PEDF’s neurotrophic activity is mediated by interactions with cell surfaces. Focusing on retinoblastoma and cerebellar granule cells, we prospected for PEDF receptors and found evidence for 1) a saturable, specific, and high-affinity class of receptors on the surface of both cells, with characteristics of an 80-kDa plasma membrane protein, and 2) the amino-terminal region in PEDF that interacts with the receptor. Furthermore, using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDF-binding partner, which we term PEDF-R. We studied PEDF-R as a receptor for PEDF and found that the PEDF-R polypeptide has specific and high binding affinity for PEDF, has a potent phospholipase A(2) activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. Our work, the first step in the biological activity of PEDF is the binding to receptors on the surface of target cells, i.e., PEDF-R, a phospholipase-linked membrane protein with high affinity for PEDF, suggests a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal, a significant advance in the elucidation of PEDF’s mechanism of action.
We also investigated the association of PEDF with extracellular matrixes (ECM) and found that PEDF can interact with glycosaminoglycans and collagens. In the folded PEDF protein, the binding sites for these two ECM components are distinct and separated from each other, from the binding site for the receptor and from the serpin exposed loop. Our functional studies showed that glycosaminoglycans can act as positive modulators of the PEDF-receptor interactions. These findings are significant because the association of PEDF with ECMs may be used in vivo to regulate this protein, i.e., by spatial and temporal localization and/or controlling its activities.
We also studied the effects of matrix metalloproteinases (MMP) type-2 and type-9 on PEDF and demonstrated that they can fully degrade PEDF in a calcium dependent-fashion, abolishing its neurotrophic and antiangiogenic activities. Moreover, we found that hypoxia and VEGF can decrease PEDF protein levels by stimulating the MMP-mediated proteolytic degradation of PEDF. Interestingly, the expression and secretion of MMP-2/-9 and of VEGF correlate positively with the progression of neovascular ocular diseases, while the levels of PEDF correlate inversely. Most importantly, these results reveal a novel post-translational mechanism for downregulating PEDF by MMP-mediated proteolytic degradation that explains hypoxia-provoked increases in the VEGF/PEDF ratio, angiogenesis and/or neuronal death.
In preparation for protein therapeutics, we explored direct delivery of the PEDF protein to the choroid and retina. We demonstrated the permeability properties of the sclera and choroid/RPE to PEDF. Upon subconjunctival delivery, this relatively large protein traversed the sclera-choroid-RPE layers to reach the retina. Our conclusions suggest that subconjunctival routes of PEDF protein delivery offer novel, feasible and minimally invasive means for administration in the clinic.
We hope our PEDF research lays the groundwork for the development of therapies for diseases involving defective neuronal differentiation or cell survival, such as retinitis pigmentosa, age-related macular degeneration, and amyotrophic lateral sclerosis. The reported antiangiogenic effects of PEDF suggest it may also be useful in treating diseases in which new blood vessel formation plays a role, such as diabetic retinopathy, age-related macular degeneration, tumor growth, and rheumatoid arthritis.
The role of a cell-surface receptor in the mechanisms of action of PEDF represents a key aspect of regulation. Along these lines, our studies focus on structure-function relationships and enzymatic characterization of PEDF receptors, and their distribution in the normal and diseased retina. Consistent with our goal of elucidating the mechanisms of action of PEDF, we are interested in signal transduction and the expression of genes affected by PEDF’s stimuli. Given that protein therapeutics pose great challenges in development, production, delivery, safety, and stability, future work will be directed toward the molecular design of potent PEDF peptides for ocular treatment to achieve altered binding specificity and prevention of side effects, and the development of sustained delivery systems for PEDF in animal models of retinal and CNS diseases.
|S. Patricia Becerra
|Monika Deshpande||IRTA Fellow (VP)||firstname.lastname@example.org|
|Sylvia Locatelli-Hoops||IRTA Fellow (VP)||email@example.com|
|Preeti Subramanian||IRTA Fellow (VP)||firstname.lastname@example.org|
Samtani S, Amaral J, Campos MM, Fariss R, Becerra SP.: Doxycycline-mediated Inhibition of Choroidal Neovascularization. Invest Ophthalmol Vis Sci. 2009 Jun 10, 2009. PubMed
Becerra SP, Perez-Mediavilla LA, Weldon JE, Locatelli-Hoops S, Senanayake P, Notari L, Notario V, Hollyfield JG.: Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site. J Biol Chem. 283(48):33310-20, 2008. Epub 2008 Sep 19. PubMed
Perruccio EM, Rowlette LL, Comes N, Locatelli-Hoops S, Notari L, Becerra SP, Borrs T.: Dexamethasone increases pigment epithelium-derived factor in perfused human eyes. Curr Eye Res. 33(5):507-15, 2008. PubMed
Lilja AE, Chang WL, Barry PA, Becerra SP, Shenk TE.: Functional genetic analysis of rhesus cytomegalovirus: Rh01 is an epithelial cell tropism factor. J Virol. 82(5):2170-81, 2008. Epub 2007 Dec 19. PubMed
Notari, L, Baladron, V., Aroca-Aguilar, J.D., Balko, N., Heredia, R., Meyer, C., Notario, P.M., Saravanamuthu, S., Nueda, M.L., Sanchez-Sanchez, F., Escribano, J., Laborda, J., Becerra, S.P.: Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor. J. Biol. Chem. 281,38022-38037, 2006. PubMed
Amaral, J., Campos, M.M., Becerra, S.P., Fariss, R.N.: A novel imaging technique for experimental choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 47, 5163-5170, 2006. PubMed
Becerra, S.P.: Focus on Molecules: Pigment epithelium-derived factor (PEDF). Exp. Eye Res. 82:739-40, 2006. PubMed
Amaral, J., Fariss, R.N, Campos,M.M., Robison, Jr,W.G., Kim,H., Lutz, R., Becerra, S.P.: Transscleral-RPE permeability of PEDF and ovalbumin proteins: implications for subconjunctival protein delivery. Invest. Ophthalmol. Vis. Sci. 46, 4383-92, 2005. PubMed
Notari, L., Miller, A., Martnez, A., Amaral, J., Ju, M., Robinson, G., Smith, L.E.H., Becerra, S.P.: Pigment epithelium-derived factor is a substrate for matrix metalloproteinase type 2 and type 9: implications for downregulation in hypoxia. Invest. Ophthalmol. Vis. Sci. 46, 2736-2747, 2005. PubMed
Becerra, S.P., Fariss, R.N., Wu, Y.-Q., Montuenga, L., Wong, P., Pfeffer, B.A.: Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor matrix: apical secretion and distribution. Exp Eye Res. 78, 223-234, 2004. PubMed
Alberdi, E.M., Weldon, J.E., Becerra, S.P.: Glycosaminoglycans in human retinoblastoma cells: Heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions. BMC Biochemistry 4:1, 2003 (19 February 2003) PubMed
Becerra, S.P., Amaral, J.: Erythropoietin–an endogenous retinal survival factor. N. Engl. J. Med. 347, 1968-1970, 2002. Review. PubMed
Meyer, C., Notari, L., and Becerra, S.P.: Mapping the type I collagen-binding site on pigment epithelium-derived factor. Implications for its antiangiogenic activity. J. Biol Chem. 277, 45400-45407, 2002. PubMed
Bilak, S.R., Bilak, M.M., Corse, A.M., Royal, W., Becerra, S.P., Kuncl, R.W.: Pigment Epithelium-Derived Factor is Elevated in CSF of Patients with ALS. J. Neurochem. 81, 178-184, 2002 PubMed
Gao, G., Li, Y., Fant, J., Crosson, C.E., Becerra, S.P., and Ma J.X.: Difference in ischemic regulation of vascular endothelial growth factor and pigment epithelium–derived factor in brown norway and sprague dawley rats contributing to different susceptibilities to retinal neovascularization. Diabetes 51, 1218-25, 2002 PubMed
Kuncl, R.W., Bilak, M.M., Bilak, S.R., Corse, A.M., Royal, W., Becerra, S.P.: Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis. J. Neurochem. 81, 178-184, 2002 PubMed
Aymerich, M.S., Alberdi, E., Martinez, A., and Becerra, S.P.: Evidence for pigment epithelium-derived factor (PEDF) receptors in the neural retina. Invest. Ophthalmol. Vis. Sci. 42, 3287-3293, 2001 PubMed
Nomura, T., Yabe, T. Mochizuki, H., Reiser, J., Becerra, S.P. and Schwartz, J.P.: Survival effects of pigment epithelium-derived factor (PEDF) expressed by a lentiviral vector in rat cerebellar granule cells. Dev. Neurosci. 23, 145-52, 2001 PubMed
Cayouette, M., Smith, S.B., Becerra, S.P., and Gravel, C.: Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations Neurobiol. Disease 6, 523-532, 1999 PubMed
Alberdi, E., Aymerich, M.S., and Becerra, S.P.: Binding of pigment epithelium-derived factor (PEDF) to retinoblastoma and cerebellar granule cells: Evidence for a PEDF receptor J. Biol. Chem. 274, 31605-31612, 1999 PubMed
Houenou, L.J., D=Costa, A.P., Li, L., Turgeon, V.L., Enyadike, C., Alberdi, E., and Becerra, S.P.: Pigment epithelium-derived factor (PEDF) promotes the survival and differentiation of developing spinal motor neurons J. Comparative Neurology 412, 506-514, 1999. PubMed
Alberdi, E., Hyde, C.C., and Becerra, S.P.: Pigment epithelium-derived factor binds to glycosaminoglycans: Analysis of binding site. Biochemistry 37, 10643-10652, 1998 PubMed
Perez-Mediavilla, L.A., Chew, C., Campochiaro, P.A., Nickells, R.W., Notario, V., Zack, D.J. and Becerra, S.P. Sequence and expression analysis of bovine pigment epithelium-derived factor (PEDF). Biochim. Biophys. Acta 1398, 203-214, 1998 PubMed
Araki, T., Taniwaki, T., Becerra, S.P., Chader, G.J., and Schwartz, J.P.: Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death. J. Neurosci. Res. 53, 7-15, 1998 PubMed
Sugita, Y., Becerra, S.P., Chader, G.J., and Schwartz, J.P.: Pigment epithelium-derived factor (PEDF) has direct effects on microglia and indirect effects on astrocytes in culture. J. Neurosci. Res. 49, 710-718, 1997 PubMed
Taniwaki, T., Hirashima, N., Becerra, S.P., Chader, G.J., Etcheberrigaray, R., and Schwartz, J.P.: Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induce neurotoxicity. J. Neurochem. 68, 26-32, 1997 PubMed
Becerra, S.P.: Structure-function relationships of PEDF: A noninhibitory serpin with neurotrophic activity. Adv. Exp. Med. Biol. 425, 223-237, 1997 PubMed