Uncovering features that control selective protein glycation.
Sjoblom, Nicole.
2019
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Posttranslational
modifications (PTMs) are covalent alterations of proteins following host cell synthesis
and they have key functional roles implicated in several diseases. In this work, we
examine two unique and understudied PTMs: glycation and bacterial effector-mediated
phosphoribosylation. Glycation, the spontaneous addition of reducing sugars or sugar
metabolites onto proteins, has been ... read moreconnected to multiple disorders including diabetes,
cancer, and other age-related diseases. While several studies have established that
glycation is a selective reaction, meaning that glycation occurs preferentially on a
protein, there is no current understanding for what controls the susceptibility of
certain proteins to become glycated. Therefore, we report one of the first studies to
experimentally validate rules that govern selective protein glycation. Paired with mass
spectrometry, we screened a panel of proteins in vitro with methylglyoxal to identify 80
unique sites of glycation and correlate these data with chemical and structural
features. We determine that the extent of glycation does not correlate with predicted
pKa values at reactive sites. Moreover, our data reveal that primary sequence dictates
the likelihood that site will become glycated, while nearby structure further shapes the
glycation outcome. We determine that nearby acidic residues hinder glycation, whereas a
combination of Tyr and polar residues promote glycation reactivity. To expand upon our
established research in a biological setting, we studied the selective modification of
ubiquitin. Previous work has led to the hypothesis that modified ubiquitin impairs the
ubiquitin-proteasome system. Therefore, we performed a focused review of the selective
glycation of ubiquitin in vitro and in vivo. Through time course studies and examination
with additional reactive aldehydes, we determine that Arg42 is the most reactive site
for protein glycation for methylglyoxal and glyoxal, two potent dicarbonyls. Moreover,
we demonstrate that ubiquitin can be glycated in HEK293T cells linking glycation to its
functional effects in the ubiquitin-proteasome system. Herein, we also examine the
reactivity of ubiquitin towards the bacterial effector protein SdeC from Legionella
pneumophila. SdeC is involved in host endoplasmic reticulum rearrangements during
infection by L. pneumophila. Utilizing mass spectrometry, we examine the nature of the
ubiquitin modifications made by SdeC. We show that SdeC catalyzes a multi-step
modification resulting in phosphoribosylation on Arg42. Moreover, we reveal that SdeC
can catalyze a unique crosslink formation with HA-tagged ubiquitin, through a ribose
phosphate linkage between ubiquitin Arg42 and HA-tag Tyr2. Together, these results
demonstrate the unique reactivity of Arg42 of ubiquitin. Combined, this work surveys
protein glycation and SdeC-mediated phosphoribosylation. This study identifies key
trends in protein glycation reactivity and is one of the first reports to experimentally
confirm them. We also determine the mechanism through which SdeC catalyzes its PTM. We
show that ubiquitin Arg42 is highly reactive towards glycation and SdeC, suggesting that
this specific site of modification has functional effects, likely within the
ubiquitin-proteasome system. Therefore, this work provides critical new insights into
understudied PTMs with biological
implications.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Biochemistry.
Advisor: Rebecca Scheck.
Committee: Andrew Bohm, Joshua Kritzer, Peter Brodeur, and David Spiegel.
Keyword: Biochemistry.read less - ID:
- 8623j977j
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