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Scientific Report 2008


Maintaining the Proteome to Ameliorate Human Disease

J.W. Kelly, S. Choi, E. Culyba, M.T.A. Dendle, D. Du,
C. Fearns, A. Fuller, T.-W. Mu, A. Murray, D. Ong, J. Paulsson, E.T. Powers, P. Rao, M. Saure, R. Simkovsky, S. Siegel,
J. Solomon, K. Usui, Y. Wang, I. Yonemoto, Z. Yu

Maintenance of the proteome (proteostasis) both inside and outside human cells is essential for development, reproduction, and successful aging. Deficiencies in proteostasis lead to many metabolic, oncologic, neurodegenerative, and cardiovascular diseases. Understanding the mechanisms of proteostasis, especially defects in the pathways of the proteostasis network that occur with aging, enables the design of new therapeutic strategies to ameliorate age-onset protein misfolding diseases, a main goal of our research. We use animal and cell-based disease models and biophysical approaches in combination with medicinal chemistry and structure-based drug design. Our collaborators, W.E. Balch, Department of Cell Biology; J. Buxbaum, Department of Molecular and Experimental Medicine; J.R. Yates, Department of Chemical Physiology; E. Masliah, University of California, San Diego; and A. Dillin, the Salk Institute for Biological Studies, La Jolla, California, play an essential role in our multidisciplinary approach.

Amelioration Of Lysosomal Storage Diseases

Lysosomal storage diseases are loss-of-function diseases often caused by a mutation in one of the lysosomal enzymes, which results in excessive misfolding of the enzyme within the endoplasmic reticulum and cytosolic degradation instead of proper folding and trafficking of the enzyme to the lysosome. In 2 separate studies, we found that the innate proteostasis capacity of a cell can be enhanced with small molecules termed proteostasis regulators to fold mutated enzymes that would otherwise misfold and be degraded, resulting in increased trafficking of the mutated enzyme to the lysosome and increased function. In the first study, we found that diltiazem and verapamil, L-type calcium channel blocker drugs approved by the Food and Drug Administration, increased folding capacity in the endoplasmic reticulum, trafficking, and activity of mutant lysosomal enzymes associated with 3 distinct lysosomal storage diseases: Gaucher disease, α-mannosidosis, and type IIIA mucopolysaccharidosis. These compounds likely act by calcium ion—mediated enhancement of endoplasmic reticulum lumenal chaperone function. In the second study, we discovered that 2 proteostasis regulators partially restored folding, trafficking, and function of mutant enzymes in Gaucher and Tay-Sachs cell lines by activating the unfolded protein response, a signaling pathway that influences proteostasis in the secretory pathway. Moreover, we found that the combination of a proteostasis regulator and a pharmacologic chaperone, a chemical that binds directly to a given enzyme and thereby stabilizes the enzyme, synergistically restored enzyme function, because of their distinct mechanisms of action.

Understanding The Etiology Of Alzheimer's Disease

We are interested in understanding the molecular and mechanistic basis for the age-onset nature of Alzheimer's disease. Genetic and biochemical evidence implicates aggregation of amyloid β-peptide (Aβ>), enabled by an age-onset decrease in proteostatic capacity, as the cause of neurodegeneration in this disease; however, precise identification of the toxic structure and the mechanism of neurotoxic effects remain elusive. In patients with Alzheimer's disease, the correlation between disease severity and the concentration of spherical aggregates, annular structures, protofibrils and other soluble oligomeric species is better than the correlation between disease severity and the concentration of fibrillar amyloid.

Previously, we showed that mutating the phenylalanine 19—phenylalanine 20 backbone amide bond to an E-olefin bond allows the formation of spherical aggregates to the exclusion of fibrils. In a more extensive amide-to-ester mutagenesis scan through the hydrophobic core (residues 17—21) of Aβ 1-40, we compared the mutants with wild-type Aβ 1-40 and the E-olefin Aβ 1-40 mutant. Even though the E-olefin mutant, the amide-to-ester mutant, and wild-type Aβ 1-40 form aggregates of different morphologies, all 3 types of aggregates were similarly toxic to PC12 neuronal cells. This finding suggests that a common, but low-abundance, aggregate morphology mediates toxic effects or that several different aggregate morphologies are similarly toxic.

Oxidized Metabolite Enhancement Of Amyloid Formation

One of the central mysteries of Alzheimer's disease is how Aβ forms amyloid in vivo when both thermodynamic and kinetic barriers against aggregation exist. We propose that covalent modification of Aβ by small-molecule oxidation products can explain, at least in part, the ability of Aβ to form amyloid at physiologic concentrations and thus place a load on the proteostasis network. Using Aβ conjugates site-specifically modified with a cholesterol aldehyde at aspartic acid 1, lysine 16, or lysine 28, we found that modification lowered the critical concentration for aggregation into the nanomolar range, within the physiologic concentration range of Aβ and dramatically increased the rate of aggregation. Aβ modified at lysine 16 formed amorphous aggregates fastest and at the lowest concentrations (within 2 hours at 20 nM).

The same cholesterol aldehyde is found in human atherosclerotic lesions and rapidly promotes apolipoprotein C-II amyloid formation in vitro. Thus, enhancement of amyloid formation by oxidized metabolites appears to be common to several diseases and suggests that strategies to prevent such modification may have therapeutic potential for a spectrum of human diseases.

Treating Transthyretin Amyloidogenesis

Transthyretin is 1 of 27 secreted human proteins, including amyloid, known to misfold and misassemble into extracellular aggregates. The rate-limiting step in amyloid formation by transthyretin is the dissociation of the tetramer. We have developed kinetic stabilizers of the tetrameric structure of transthyretin that have novel chemistries and mechanisms of actions. To improve inhibitors of transthyretin amyloidogenesis, we are optimizing each of the 3 substructural elements that make up a typical inhibitor: the 2 aryl rings and the linker that joins the rings. We evaluated structural modifications to the aryl ring 1 by screening a library of 2-arylbenzoxazoles that have thyroid hormone—like aryl substituents on the 2-aryl ring. The 3,4,5-substituted thyroxine-like aryl ring appears to be the optimal solution for the structure of aryl ring 1. In addition, we synthesized 40 bisaryl compounds to optimize the structure of the linker. We found that direct connection of the 2 aryls, or linkage through nonpolar E-olefin or —CH2CH2— substructures, generates the most potent and selective inhibitors of transthyretin amyloidogenesis. Five high-resolution (1.4—1.8 Å) x-ray crystallography structures of transthyretin reveal that the 3,5-dimethyl-4-hydroxyphenyl ring preferentially occupies the inner cavity of the thyroxine-binding site and that the 3,5-dibromo-4-hydroxyphenyl aryl prefers the outer cavity because the phenol is deprotonated with flanking electron withdrawing group substitution. A study to optimize the remaining aryl ring is well under way.

Tetramers of transthyretin can also be kinetically stabilized by trans-suppression, as we showed previously with T119M transthyretin subunit incorporation into the tetramer, which stabilizes heterotetramers containing T119M and V30M transthyretin subunits. In an analogous manner, heterotetramers composed of murine transthyretin and human transthyretin subunits are kinetically stable and nonamyloidogenic. This information is important for evaluating transgenic models of human transthyretin amyloidosis in which the transgenic animals have a low copy number of the mutant amyloidogenic human transthyretin gene.

Understanding And Ameliorating Gelsolin Amyloidosis

Gelsolin amyloid disease is another age-onset degenerative malady linked to protein aggregation that is thought to be due to an age-associated decline in proteostasis. A mutation in gelsolin (D187N) leads to aberrant folding and cleavage by furin within the Golgi apparatus during trafficking. Subsequent cleavage of the gelsolin fragment by the matrix metalloprotease MT1-MMP outside the cell results in 5- and 8-kD fragments of gelsolin, which deposit as amyloid in the extracellular matrix. In collaboration with W.E. Balch, Department of Cell Biology, we have developed 2 transgenic mouse models of human D187N gelsolin amyloidosis that recapitulate the aberrant endoproteolytic cascade and the aging-associated decline in proteostasis that result in extracellular amyloidogenesis in humans.


Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. Adapting proteostasis for disease intervention. Science 319:916, 2008.

Bieschke, J., Siegel, S.J., Fu, J., Kelly, J.W. Alzheimer's Aβ peptides containing an isostructural backbone mutation afford distinct aggregate morphologies but analogous cytotoxicity: evidence for a common low-abundance toxic structure(s)? Biochemistry 47:50, 2008.

Dillin, A., Kelly, J.W. The yin-yang of sirtuins. Science 317:461, 2007.

Fowler, D.M., Koulov, A.V., Balch, W.E., Kelly, J.W. Functional amyloid: from bacteria to humans. Trends Biochem. Sci. 32:217, 2007.

Jäger, M., Dendle, M., Kelly, J.W. A cross-strand Trp-Trp pair stabilizes a WW domain at the expense of function. Protein Sci. 16:2306 2007.

Jäger, M., Nguyen, H., Dendle, M., Gruebele, M., Kelly, J.W. Influence of hPin1 WW N-terminal domain boundaries on function, protein stability, and folding. Protein Sci. 16:1495, 2007.

Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008.

Kelly, J.W. Compromised cellular folding fidelity results in numerous clinically important diseases. Nature 446:112, 2007.

Liu, F., Du, D., Fuller, A.A., Davoren, J.E., Wipf, P., Kelly, J.W., Gruebele, M. An experimental survey of the transition between two-state and downhill protein folding scenarios. Proc. Natl. Acad. Sci. U. S. A. 105:2369, 2008.

Mu, T.-W., Fowler, D.M., Kelly, J.W. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis. PloS Biol. 6:e26, 2008.

Münch, J., Rücker, E., Ständker, L., Adermann, K., Goffinet, C., Schindler, M., Wildum, S., Chinnadurai, R., Rajan, D., Specht, A., Giménez-Gallego, G., Sánchez, P.C., Fowler, D.M., Koulov, A., Kelly, J.W., Mothes, W., Grivel, J.C., Margolis, L., Keppler, O.T., Forssmann, W.G., Kirchhoff, F. Semen-derived amyloidogenic prostatic acidic phosphatase fragments dramatically enhance HIV infection. Cell 131:1059, 2007.

Reixach, N., Foss, T.R., Santelli, E., Pascual, J., Kelly, J.W. Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic: a lesson in the generation of transgenic models of diseases involving oligomeric proteins. J. Biol. Chem. 283:2098, 2008.

Stewart, C.R., Wilson, L.M., Zhang, Q., Pham, C.L.L., Waddington, L.J., Staples, M.K., Stapleton, D., Kelly, J.W., Howlett, G.J. Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation. Biochemistry 46:5552, 2007.

Wiseman, R.L., Koulov, A., Powers, E.T., Kelly, J.W., Balch, W.E. Protein energetics in maturation of the early secretory pathway. Curr. Opin. Cell Biol. 19:359, 2007.

Wiseman, R.L., Powers, E.T., Buxbaum, J.N., Kelly, J.W., Balch, W.E. An adaptable standard for protein export from the endoplasmic reticulum. Cell 131:809, 2007.

Yu, Z., Sawkar, A.R., Kelly, J.W. Pharmacologic chaperoning as a strategy to treat Gaucher disease. FEBS Lett. 274:4944, 2007.


Jeffery W. Kelly, Ph.D.
Chairman, Molecular and Experimental Medicine

Evan Powers, Ph.D.
Assistant Professor

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