Scientific Report 2008
Maintaining the Proteome to Ameliorate
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
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
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
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
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
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 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
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.
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,
Yu, Z., Sawkar, A.R., Kelly, J.W.
as a strategy to treat Gaucher disease. FEBS Lett. 274:4944, 2007.