Alzheimer’s disease, brain neurons become clogged with tangled
proteins. Scientists suspect these tangles arise partly due to
malfunctions in a little-known regulatory system within cells. Now,
researchers have dramatically increased what they know about this
particular regulatory system in mice. Such information will help
scientists better understand Alzheimer’s and other diseases in humans
and could eventually provide new targets for therapies.
a study released online in the Proceedings of the National Academy of
Sciences Early Edition this week, the team at least doubled the number
of proteins found to be subject to a type of regulation based on a sugar
known as O-GlcNAc (oh-GLIK-nak). The O-GlcNAc
system likely adds another layer of control to the proteins that serve
as a brain cell’s widgets and gears—control that might be muddled in
Alzheimer’s brains known to have problems in sugar metabolism.
found many novel proteins providing insights into new aspects of cell
biology,” said analytical biochemist Feng Yang of the Department of
Energy’s Pacific Northwest National Laboratory and lead author on the
study. “We think O-GlcNAc is fine-tuning cellular processes.”
In addition to finding hundreds of proteins modified by O-GlcNAc, the team found that almost all the O-GlcNAc
proteins were also subject to the most common form of protein
regulation, which uses small phosphate molecules to turn proteins on and
off. This suggests a larger coordination between the two regulatory
results show there’s a level of complexity about how biology operates
that we’ve been largely blind to,” said PNNL’s Richard D. Smith, who
leads the proteomics team at PNNL. Proteomics researchers try to
understand how a cell functions based on the numbers and types of its
proteins at work, which are collectively known as the proteome
during the Human Genome Project, we asked, how could so few genes
produce the complexity of an organism or even a single cell, and how
could minor variations in our DNA explain the diversity we see all
around us? Clearly the proteome is the answer,” said Smith.
are the tools, gears and gadgets that run a cell. Regulatory systems
within cells turn proteins on and off by attaching or detaching small
molecules to the proteins, like a switch. The most common switch
involves adding or removing phosphates, and biologists have known for a
long time that these switches can run amiss in cancer and other
diseases. Drugs affect players in the phosphate regulatory system to try
to fix the errors.
A couple decades ago, researchers found that O-GlcNAc, a kind of sugar, could also work like a switch, turning proteins on or off. Scientists found proteins decorated by O-GlcNAc, as well as other proteins that attach or remove the sugar—all essential parts to the system.
But they had trouble finding enough O-GlcNAc
proteins to get the whole story. Few proteins bore the small sugar, and
those that did tended to lose the accessory while being manhandled in
the lab. Researchers could make up for some of these problems by
starting with more tissue or cultured cells, but they knew if they
wanted to look for these modifications in real-life scenarios such as
clinical samples, they would need to be able to find the sugar with a
small amount of starting material.
overcome these difficulties, Smith, Yang and their colleagues at PNNL
and four research institutions combined their expertise in the O-GlcNAc
system with instruments developed at EMSL, DOE’s Environmental
Molecular Sciences Laboratory on the PNNL campus. First they improved
how they purified protein from mouse brain tissue to reinforce the sugar
attached to proteins. Then they used instruments that excelled at
detecting rare proteins in small samples.
addition, they looked for the sugar-dotted proteins in mouse brain
samples from engineered animals that had a mouse version of Alzheimer’s.
These mice make too much of three key proteins implicated in
Alzheimer’s disease in people, including the Tau protein, which forms
the hallmark tangles in brain neurons.
Pack o’ proteins
To test how well their methods found O-GlcNAc
proteins, the PNNL-led team started with tissue from either healthy or
diseased mouse brain tissue. From the healthy tissue, the team found
274 different proteins marked with O-GlcNAc.
Many of them sported more than one sugar molecule, because the team
found a total of 458 attachment sites on those 274 proteins—triple the
number of sites found in any previous study. The large number of sites
allowed the team to identify similarities between O-GlcNAc sites, as well as O-GlcNAc sites on previously unexplored proteins.
Of the 274 O-GlcNAc
proteins, 106 had already been identified in other studies. These
proteins held a variety of jobs, including forming part of a cell’s
scaffolding, or in nerve growth or in other nerve-related occupations
such as learning and memory.
left 168 newly-identified proteins. Based on what the proteins looked
like, the team classified most of them as likely being involved in cell
signaling, regulating how genes are expressed, or, again, in cell
The PNNL-led team then looked at the proteins found in the Alzheimer’s-like mouse brain. They found about a third fewer O-GlcNAc-marked proteins. That result also supports earlier work that suggested there is damaged O-GlcNAc regulation in Alzheimer’s brains in people.
Fraternizing phosphates and other biology
of the more exciting things the researchers found had to do with the
most common regulatory system in cells, the phosphate system. More than
98% of the O-GlcNAc
proteins also had sites that would accept a phosphate, suggesting those
proteins are also under the control of that system.
And about a quarter of the O-GlcNAc
sites were close enough to the phosphate sites to interfere with that
switch, suggesting cross-talk between the two types of regulation. A
phosphate is smaller than O-GlcNAc
and has a strong negative electrical charge. The sugar is neutral but
bulkier. Those characteristics could have different effects on the
structure of the protein and greatly increases the range of possible
biological effects due to the complexity of the combined switching
Lastly, until this study, most of the proteins known to be under O-GlcNAc
control largely live their lives within the cells. But the PNNL-led
team found a half-dozen proteins that had to be controlled by O-GlcNAc outside a cell, based on where their O-GlcNAc site fell on the body of the protein.
Now, the team is planning to measure both regulatory systems in concert.
revealing to see how many proteins are modified. If we’re going to
understand biological systems, we need to understand the interplay of
the different types of modifications,” said Smith.
contributing to this work came from PNNL, New York State Institute for
Basic Research in Developmental Disabilities, Staten Island, N.Y., Johns
Hopkins University School of Medicine, Baltimore, Md., the University
of Virginia, Charlottesville, Va. and Albert Einstein College of
Medicine in New York City, N.Y.
work was supported by PNNL, EMSL and the National Institutes of
Health’s National Center for Research Resources and National Institute
of General Medical Sciences.
Tandem Mass Spectrometry identifies many mouse brain O-GlcNAcylated proteins including targets of an EGF domain-specific OGT