The Non-Enzymatic Glycation of Ubiquitin: A Structural and Functional Study Mark Esposito and Roger Sandwick Department of Chemistry and Biochemistry Middlebury College, Middlebury, Vt. 05753

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The Non-Enzymatic Glycation of Ubiquitin A
Structural and Functional StudyMark Esposito and
Roger SandwickDepartment of Chemistry and
Biochemistry Middlebury College, Middlebury, Vt.
05753

• Monitoring the Ubiquitination/ Proteolysis of
  Yeast cytochrome c
• Figure 2. Extracted ion chromatogram for heme
  group of urea-arrested proteolysis of cytochrome
  c with wild-type ubiquitin. A) 0 minute arrest,
  integration yields 0 MIU. B) 2.5 minute arrest,
  integration yields 24.2 MIU. C) 5 minute arrest,
  integration yields 84.6 MIU.
• Figure 3. Extracted ion chromatogram for heme
  group of urea-arrested proteolysis of cytochrome
  c with R5P-glycated ubiquitin A) 0 minute arrest,
  integration yields 49.9 MIU. B) 2.5 minute
  arrest, integration yields 68.7 MIU. C) 5 minute
  arrest, integration yields 54.2 MIU

• Abstract
• The glycation of ubiquitin with ribose
  5-phosphate (R5P) and glucose was studied to
  determine the effect of post-translationally
  modified ubiquitin on intracellular proteolysis.
  Intracellular proteolysis is initiated by the
  conjugation of ubiquitin to protein substrates
  via an ensemble of enzymes to 1) form a thiol
  ester bond to the C-terminus of ubiquitin, 2)
  transport conjugated ubiquitin to target protein
  substrates and 3) ligate ubiquitin to surface
  e-amino groups of a target protein substrate.
  Polyubiquitination then can occur in a processive
  manner through e-amino groups on ubiquitin. Our
  investigations focused on identifying the
  location of glycation sites on the ubiquitin
  protein and on developing a method for assessing
  the effect that glycation has on ubiquitin
  activity. R5P-glycated and glucose-glycated
  ubiquitin isolated by boronate and anion exchange
  chromatography showed as many as three glycation
  events within a 72 h period. Trypsin digestion
  studies suggest glycation occurs preferentially
  at certain e-amino groups. A novel method which
  employs yeast cytochrome c as a ubiquitination
  target substrate and LC-MS for subsequent
  analysis is under development for use in
  assessing the functionality of modified
  ubiquitin. Preliminary results suggest that this
  is a robust method for the detection of
  ubiquitination. Further refinement of this method
  is necessary before the effects of glycation on
  ubiquitination can be analyzed.

A
B
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B
Introduction The intracellular proteolytic
degradation of short-lived and abnormal proteins
is achieved through the conjugation and ligation
of ubiquitin to protein substrates. Ubiquitin is
a 8.6 kDa protein highly conserved throughout the
entire phylogenic tree. The biochemical pathway
resulting in covalent ligation is illustrated
below. (Ciechanover and Hersko, Annu. Rev.
Biochem. 1998) The crucial importance of
lysine residues in both initial ubiquitin
ligation and subsequent polyubiquitination events
makes non-enzymatic glycation of proteins a
pertinent field of study. It has been previously
established that glycation of protein substrates
inhibits proteolysis, however the effects of
glycation on the ubiquitin pathway have not yet
been explored. Generally, the glycation of
e-amino groups is depicted below. (Watkins et.
al., J. Bio. Chem. 1985 )
C
Results Purification of Glycated
Ubiquitin Figure 1. A) Sample
incubated at 37 C for 72 hr at 1 M glucose and 1
mg/mL Ubiquitin. B) Glucose incubated ubiquitin
purified through boronate chromatography. C)
Sample incubated at 37 C for 24 hr at 0.1 M R5P
and 1 mg/mL Ubiquitin. D) R5P incubated sample
purified through anion exchange
chromatography. Trypsin Digest of Glycated
Ubiquitin Enrichment Table 1. Trypsinated
fragment sequence and LC-MS analysis of
trypsinated, R5P-glycated ubiquitin. Percent
fragment present obtained by comparison to
control integrations and reference to fragment
10.
A
B
C
D
E
F
Objectives 1) In order to hypothesize the
effects of glycation, the preferential sites of
glycation must be established. 2) Though very
sensitive, currents methods for examining
ubiquitin mediated degradation through
radioactive counting are impractical for rapidly
examining the effects of proteolysis. Thus a
novel method for monitoring proteolysis will be
developed. 3) To quantify the effects of
glycation, a population of ubiquitin glycated by
either R5P or glucose must be purified. Methods
will be developed for the purification of both
species of modified ubiquitin. 4) Purification
and proteolysis methods will be synthesized to
determine the effects of glycation on ubiquitin
mediated proteolysis.
Methods Ubiquitin Glycation and Purification
Ubiquitin was reacted with R5P or glucose for 24
- 72 hours. Glucose glycated samples were
purified through boronate affinity
chromatography. R5P glycated samples were
purified through anion exchange chromatography.
Relative concentration was established by the BCA
assay. Glycation Analysis Purified samples were
subjected to trypsin digest and analyzed by
LC-MS. Development of Novel Proteolysis Method
Bovine erythrocytes were obtained from Vermont
Livestock S P. Cells were depleted of ATP and
lysed with DTT. The ensemble of enzymes necessary
for proteolysis were purified through ion
exchange chromatography. Reactions buffered at
low ionic strength containing ubiquitin, ATP, an
ATP regeneration system , purified erythrocyte
fractions, and yeast cytochrome c were arrested
with urea and monitored at varying time scales
with LC-MS. Heme growth and UV absorbance were
quantified to establish the extent of
proteolysis. Purified glycated ubiquitin was used
in conjunction with proteolysis reagents to
assess effects of glycation.
Fragment Fragment Sequence Percent Fragment Present
1 MQIFVK 18.4
2 TLTGK 6.1
3 TITLEVEPSDTIENVK 33.9
4 AK N/A
5 IQDK 12.4
6 EGIPPDQQR 20.9
7 LIFAGK 68.4
8 QLEDGR 9.7
9 TLSDYNIQK 7.6
10 ESTLHLVLR 100.0
11 LR N/A
12 GG N/A