Biovetenskaper och näringslära / Biosciences and Nutrition
Abstract
Thioredoxins (Trx) are a class of small multifunctional 12-kDa proteins
that are characterized by the redox active site Trp-Cys-Gly-Pro-Cys
(WCGPC). In the oxidized (inactive) form of Trx (Trx-S2), the two
cysteines at the active site form a disulfide bond. This can then be
reduced by thioredoxin reductase (TrxR) and NADPH, the so-called
thioredoxin system, to a dithiol (Trx- (SH)2). which can then act as a
general protein disulfide reductase. Tbioredoxins are present in all
living organisms and have been isolated and characterized from a wide
variety of prokaryotic and eukaryotic cells. In this thesis we describe
the identification and functional characterization of novel members of
the thioredoxin superfamily.
We present evidence for a novel Trx (Trx2) in Escherichia coli. The E.
coli Trx2 contains two domains: an N-terminal domain of 32 amino acids
containing two CXXC motifs and a C-terminal domain with high homology to
the prokaryotic thioredoxins, containing the conserved active site. Trx2
together with TrxR and NADPH can reduce ribonucleotide reductase as well
as the interchain disulfide bridges of insulin.
Tbioredoxins are ubiquitously expressed in an tissues within the same
organism. We have identified the first tissue specific Trx (Sptrx1)
exclusively expressed in human spermatozoa. Sptrx1 is an active
thioredoxin which under native conditions appears to have a multimeric
structure.
We also identify and characterize a complete thioredoxin system (Trx2,
TrxR2) located in mitochondria. We show that Trx2 overexpressing cells
have a higher mitochondrial membrane potential that is dependent on the
function of the ATP synthase complex. Furthermore, overexpression of Trx2
was found to protect cells against the cytotoxic effects of etoposide, a
drug commonly used in anticancer treatment.
In addition, we showed that the second compound of the mitochondrial
thioredoxin system, TrxR2, is capable of reducing cytochrome c and could
protect cells against the cytotoxic effects of antimycin and myxothiazol,
chemicals that inhibit the function of complex III in the mitochondrial
electron transport chain.
Furthemore, we identified an alternative splicing variant of cytosolic
thioredoxin reductase (TrxR1b) that could bind to the Estrogen Receptors
(ER) alpha and beta. As a result of this binding, a distinct subnuclear
localization of TrxR1b was observed, co localizing with both ER alpha and
beta. TrxR1b can act as a coactivator and enhance the transcriptional
activity of ER in the classical activation pathway, which relies on the
binding of the ER to an ER response element on the DNA. By contrast,
TrxR1b is a co-repressor in the alternative pathway where ER activates
AP- I transcription independently of its DNA binding activity.
In summary, the results presented in this thesis give a better
understanding of Thioredoxin systems in both prokaryotes and eukaryotes,
with the introduction of new members in this redox superfamily of
proteins. This study, which shows a wide spectrum of functions for these
Thioredoxin systems in influencing various redox mechanisms and
processess in biological systems, indicates that there is still a great
deal of work yet to be done in this expanding field of research