THESIS
2016
xi, 45 pages : illustrations ; 30 cm
Abstract
It is well-known that selenocysteine (Sec) could be specifically incorporated into growing
polypeptides, as dened by the stop codon UGA together with SECIS sequence elements.
As a structural analog, however, Sec could compete for the tRNA
Cys against
cysteine inserting into protein directly in place of cysteine. This UGA-independent mis-incorporation
of Sec substituting cysteine, has yet to be studied on a proteome level, and
the features of Sec mis-incorporation sites remains unclear. This research aims to profile
the E. coli proteome for Sec mis-incorporation by using label-free quantitative proteomics
methods. Taking advantage of LC-MS based shotgun proteomics technology, a total of 44
selenopeptides have been identified from cells exposed to a high-selenium medium. Analysis...[
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It is well-known that selenocysteine (Sec) could be specifically incorporated into growing
polypeptides, as dened by the stop codon UGA together with SECIS sequence elements.
As a structural analog, however, Sec could compete for the tRNA
Cys against
cysteine inserting into protein directly in place of cysteine. This UGA-independent mis-incorporation
of Sec substituting cysteine, has yet to be studied on a proteome level, and
the features of Sec mis-incorporation sites remains unclear. This research aims to profile
the E. coli proteome for Sec mis-incorporation by using label-free quantitative proteomics
methods. Taking advantage of LC-MS based shotgun proteomics technology, a total of 44
selenopeptides have been identified from cells exposed to a high-selenium medium. Analysis
of the corresponding gene sequences for the identified selenopeptides showed that all
the replaced cysteines were encoded by UGU or UGC instead of UGA, and the SECIS
element was not found, indicating that the cysteines were replaced by Sec in an apparent
UGA-independent manner. Interestingly, the mis-incorporation sites did not distribute
randomly as expected. Across the proteome, some cysteine residues were consistently
replaced whereas other cysteines were not replaced at all. Secondary structure analysis
led to a possible explanation that mis-incorporation at critical sites caused misfolding
and degradation of proteins making it undetectable in LC-MS. Quantitative comparative
analysis between normal group and the high-selenium group revealed that the expression
levels of 320 proteins have changed significantly, 251 of which have been down-regulated.
Gene ontology and pathway analysis indicated that most of these proteins are involved in
the biosynthesis of amino acids, tRNA aminoacylation and oxidation-reduction reactions.
Proteins related to UGA-dependent incorporation of Sec were extensively down-regulated,
which implied that a high concentration of Sec possibly has an inhibitory effect on the
UGA-dependent pathway.
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