Many epidemiological
studies have linked the role of serum albumin to an important antioxidant
in the body. These studies consistently showed that reduced albumin levels
are associated with an increased mortality risk and the incidence of coronary
heart disease (CHD). The increased mortality and CHD incidence have mainly
attributed to the albumin’s various ligands binding ability, which
albumin acts as a buffering agent for toxic molecules of endogenous or
exogenous origin introduced into the circulation. Recent studies have
suggested the potential role of albumin as an effective antioxidant in
protecting many serum proteins from changes induced by oxidative stress.
Also, it has been shown that in the event of myocardial ischemia oxidative
stresses released from hypoxic heart tissue induce modification of albumin,
which can be characterized by reduced cobalt binding capacity of albumin.
However, molecular mechanism for this reduced metal binding capacity induced
by myocardial ischemia remains unknown.
We are studying the modifications of serum albumin induced by myocardial
ischemic event by using a novel protein expression system and site-directed
mutagenesis technique. Specifically, we are using a yeast protein expression
system, Pichia pastoris to produce wild type recombinant serum
albumin and its mutants with aim of evaluating the effects of particular
amino acid mutations or deletions on the specific sites on albumin to
cobalt. Furthermore, we plan to introduce oxidative modifications and
glycation of recombinantly produced albumin and its mutants to test
our hypothesis that the changes induced by ischemic situations may not
be limited to cobalt binding sites of albumin. The altered cobalt binding
capacity of ischemic albumin has proven to be useful in predicting the
early onset of myocardial ischemia. However, a physiological mechanism
for this myocardial ischemia modification of albumin has not been provided.
Our short term goal is to obtain the specific structural information
on which amino acids at the major metal binding sites of HSA will be
responsible for cobalt binding and how the modifications of these amino
acids will lead to the reduced cobalt binding affinity as seen in ischemia
modified albumin species. Our ultimate goal is to understand the nature
of ischemia modified albumin and thereby, possibly develop a better
diagnostic indicator for the early detection of myocardial ischemia,
which can be used in clinical settings.
The followings are the
list of clinical studies we have played major role and the results are
published in the peer-reviewed journals:
1. ApoE
genetic variant screening: Using human blood samples we amplified the
specific segment of genomic DNA which correspond to Apo E gene and analyzed
the DNA information using restriction enzyme and polymerase chain reaction
techniques. We identified many different types of Apo E isoforms.
2. Digoxin, a cardiac glycoside for the treatment of congestive heart
failure, interactions with human serum albumin: Studies involved the
estimation of the effects of HSA mutations on the pharmacokinetics of
digoxin in human body.
3. Familial Dysalbuminemic Hyperthyroxinemia (FDH) and thyroxine study:
Our lab identified and confirmed by site-directed mutagenesis and novel
gene expression system that FDH is caused by a single point mutation
on the genomic DNA. Also, we developed a new diagnostic method to detect
FDH using simple blood test.
4. Warfarin, an anticoagulant and HSA study: In this study we investigated
the various drugs and amino acid mutation effects on warfarin interactions
with serum albumin using fluorescence, site-directed mutagenesis, and
equilibrium dialysis.
5. Bilirubin, a toxic metabolite of heme and HSA study: We studied bilirubin
binding to HSA and showed that HSA has a dynamic, unusually flexible
high binding affinity site for bilirubin enabling HSA’s role as
detoxification agents.
6. Nitric Oxide, an important biological signaling molecule, and HSA
study: In this study we showed that by nitrosating HSA mutants Trp-214
is the primary nitrosation target in HSA and HSA plays major role in
NO metabolism.
7. Prostaglandin (PG) interconversion study: We showed that HSA stabilizes
and mediates PG interconversions that promote platelet disaggregation.
Using a model system at physiological pH, HSA and its mutants mediated
PG interconversions was investigated to understand molecular nature
of this process.
8. Cholesterol efflux study: We studied the role of HSA in cholesterol
efflux from cultured endothelial cells since cholesterol efflux plays
a central role by serving to mitigate cholesterol accumulation in extrahepatic
tissues. This study includes examination of the mechanism and identification
of the key amino acid residues of HSA involved in the efflux. HSA mediated
cholesterol efflux was compared to apolipoprotein A-1 (apo A1) and high-density
lipoprotein (HDL).
9. Glycated albumin study: In this study we investigated that the properties
of glycated albumin in diabetes patients and the effects on various
ligands binding interactions.
10. Ethanol effects on albumin study: We showed that ethanol influence
on warfarin binding to HSA alters the pharmacokinetics of anticoagulant
warfarin.
11. The effects of fatty acids on thyroxine binding to HSA. We used
site-directed mutagenesis and x-ray crystallography techniques to show
the effects of fatty acids on thyroxine binding to HSA. This study showed
that fatty acids binding to HSA dislocated thyroxine from its original
binding to the new binding site present in the crevice of HSA structure
between domains 2 and 3.
12. Myocardial Ischemic Modifications of HSA. In this study we showed
that myocardial ischemic patients exhibited unique cobalt binding capacity
changes due to the modifications of HSA. We sequenced N-terminal regions
of HSA and showed that the primary binding site of cobalt were not removed
from myocardial ischemia event. Currently, we are working on the mechanisms
of myocardial ischemia induced modifications of HSA.
 |
X-ray crystallographic Structure
of Human Serum Albumin (HSA) |
 |
Ligand
binding site I of HSA |
 |
Ligand
binding site II of HSA |
The figures were prepared with the program
MOLMOL (Koradi et al., 1996)