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FRISC: The Faculty Research Interests Science Comparator |
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Associate Professor of Biochemistry
Biological Chemistry
Office: (214) 648-2457
FAX: (214) 648-8856
Email: chuang01@utsw.swmed.edu
A primary goal of this laboratory is to
decipher the mechanism by which molecular
chaperones mediate folding and assembly of macromolecular structures. The principle of
spontaneous protein folding dictates that misfolded intermediates can escape from their
kinetic traps by thermal motion, and eventually reach the native conformation. However,
for a significant number of proteins, spontaneous folding may not occur on a biologically
meaningful time scale. This subset of proteins depend on the function of pre-existing
protein machineries, i.e. molecular chaperones to promote proper folding in an
energy-dependent manner. Many of these molecular chaperones are heat-shock proteins (Hsps)
that are highly conserved from bacteria to plants to humans. We currently focus on the
bacterial chaperonin duet GroEL / GroES and their mitochondrial homologues Hsp60 / Hsp10 ,
respectively. We are challenging the dogma that these chaperonins assist the folding of
single polypeptides only, and play no part in oligomeric protein assembly. Our recent data
show that GroEL and co-chaperonin GroES promote dissociation/reassociation cycles of an ab heterodimeric intermediate during a 2 b 2
assembly of the decarboxylase (E1) component of the human mitochondrial branched-chain a -ketoacid dehydrogenase (BCKD)
complex. The finding describes a novel role for these chaperonins in priming the assembly
intermediate for higher-order oligomerization through an iterative annealing mechanism. We
are presently dissecting interactions between the GroEL double-ring complex and the
native-like ab heterodimeric
intermediate at the three-dimensional level. As for human Hsp60, which in contrast
possesses a single-ring structure, its reaction mechanism is still poorly understood. We
are also using human E1 heterotetramers as a target protein to investigate
Hsp60/Hsp10-dependent folding and assembly both in
vitro and in vivo in a homologous system.
As a second project, we are
elucidating the biochemical mechanism underlying maple
syrup urine disease ( MSUD ) or
branched-chain ketoaciduria. MSUD is a heritable metabolic disorder in the oxidative
degradation of branched-chain amino acids leucine, isoleucine and valine, and is
manifested by often-fatal acidosis and mental retardation. The BCKD complex, which is
deficient in MSUD, is a macromolecular structure (4,000 kDa in size) organized around a
24-meric acyltransferase (E2) core, to which multiple copies of E1, a dehydrogenase (E3),
a specific kinase and a specific phosphatase are attached through ionic interactions. We
recently solved the crystal structure of the E1 heterotetramer at 2.7 Å resolution. The
information explains well the MSUD phenotype caused by mutations in E1 subunits, including
assembly defects associated with the prevalent Y393N- a substitution in homozygous
Mennonite patients. Efforts are underway to determine the structures of remaining enzyme
components of the human BCKD complex by X-ray crystallography or NMR. State of the
art molecular genetic and biochemical methods are utilized to characterize
recombinantly produced wild-type and mutant proteins. Our goal is to understand how MSUD
mutations impede catalysis, folding/assembly and protein-protein interactions between
enzyme components of the BCKD complex.
Selected Publications:
Wynn RM, Song J-L and Chuang DT (1999)
GroEL/GroES promote dissociation/reassociation cycles of a heterodimeric intermediate
during a 2 b 2
oligomeric assembly. Iterative annealing at the quaternary structure level. J Biol Chem (in
press)
Chuang JL, Wynn RM, Song J-L and
Chuang DT (1999) GroEL/GroES-dependent
reconstitution of a 2 b 2 tetramers of human mitochondrial
branched-chain a -ketoacid
decarboxylase. Obligatory interaction of chaperonins with an ab dimeric intermediate. J Biol Chem
274: 10395-10404
Huang Y-S and Chuang DT (1999) Mechanism for GroEL/GroES-mediated folding of a
large 86-kDa fusion polypeptide in vitro. J Biol
Chem 274: 10405-10412
Wynn RM, Davie, JR, Chuang JL, Cote,
CD and Chuang DT (1998) Impaired assembly of
E1 decarboxylase of the branched-chain a -ketoacid
dehydrogenase complex in type IA maple syrup urine disease. J Biol Chem 273: 3110-3118
Chuang JL, Cox RP and Chuang DT
(1997) E2 transacylase-deficient (type II) maple syrup urine disease. Aberrant splicing of
E2 mRNA caused by internal intronic deletions and association with thiamine-responsive
phenotype. J Clin Invest 100: 736-744
Chuang JL, Davie JR, Chinsky JM,
Wynn RM, Cox RP. and Chuang DT (1995) Molecular and biochemical basis of intermediate
maple syrup urine disease. Occurrence of
homozyogus G245R and F364C mutations at the E1 a locus of Hispanic-Mexican patients. J Clin Invest 95: 954-963
Wynn RM, Davie JR, Zhi W, Cox RP and
Chuang DT (1994) In vitro reconstitution of the
24-meric E2 inner-core of bovine mitochondrial branched-chain a -keto acid dehydrogenase complex:
Requirement of chaperonins GroEL and GroES. Biochemistry
33:8962-8968
Wynn RM, Davie JR, Cox RP and Chuang
DT (1992) Chaperonins GroEL and GroES promote assembly of heterotetramers ( a 2 b 2 )
of mammalian mitochondrial branched-chain a -keto acid decarboxylase in Escherichia coli. J Biol Chem 267:12400-12403
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