Scientists suspect that the reason why brain neurons become clogged with tangled proteins in Alzheimer’s disease is partially due to malfunctions in a little-known regulatory system within cells.

In a new study published online in this week’s Proceedings of the National Academy of Sciences Early Edition, researchers have made a gigantic leap forward in gaining more insight into this particular regulatory system in mice. The newly gained knowledge will provide scientists with a better understanding of Alzheimer’s and other human diseases and could eventually lead to the development of new therapies.

In their new study, the researchers discovered twice as many new proteins; these were found to to be part of a protein regulation that is based on a sugar known as O-GlcNAc (oh-GLIK-nak).

The O-GlcNAc system is thought to add another layer of control to the proteins that act as the brain’s controls, which may be confused in the brain of Alzheimer patients with known problems in metabolizing sugar.

Leading researcher, Feng Yang, an analytical biochemist of the Department of Energy’s Pacific Northwest National Laboratory said:

“We found many novel proteins providing insights into new aspects of cell biology. We think O-GlcNAc is fine-tuning cellular processes.”

Aside from discovering hundreds of proteins that were modified by O-GlcNAc, the team also established that the majority of all the O-GlcNAc proteins which belonged to the most common form of protein regulation, that uses small phosphate molecules to turn proteins on and off, which indicates that the two regulatory systems coordinate with each other.

Richard D. Smith, who leads the proteomics team at PNNL declared:

“These results show there’s a level of complexity about how biology operates that we’ve been largely blind to.”

Proteomics researchers are studying proteome (PRO-tee-ohm), i.e. cell functions based on the numbers and types of its proteins at work.

Smith continued:

“Back 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.”

Cells are controlled by proteins, which act as tools, gears and gadgets. Binding or detaching small molecules to the proteins allows regulatory systems within cells to turn proteins on and off like a switch, the most common of which is binding to or detaching phosphates. For years biologists have known that these switches can malfunction in cancer and other diseases, and drugs that influence parts in the phosphate regulatory system try repairing these errors.

About 20 years ago, researchers discovered that O-GlcNAc could also work like a switch by turning proteins on or off. They discovered proteins with O-GlcNAc attached and other proteins that bind or detach the sugar, all of which are vital parts of the system. However, they failed to find sufficient O-GlcNAc proteins to gain complete insight into the process.

They found that few proteins had O-GlcNAc attached, and those that did often lost the sugar whilst being handled in the lab. The researchers tackled part of the problem by starting with more tissue or cultured cells. However, in order to study these modifications in real-life scenarios such as clinical samples, they needed to find the sugar with a small amount of starting material.

To tackle these problems, Smith, Yang, their team at PNNL, as well as four research institutions collaborated by combining their expertise in the O-GlcNAc system with instruments developed at DOE’s Environmental Molecular Sciences Laboratory on the PNNL campus. The first step was to improve the approach of purifying the protein from mouse brain tissue, in order to reinforce the sugar attached to proteins, after which they utilized novel instruments to identify rare proteins in small samples.

They also searched for the sugar-dotted proteins in mouse brain samples from engineered animals that had a mouse version of Alzheimer’s. These mice overproduce three major proteins that occur in people with Alzheimer’s, including the Tau proteins, which produce the classic tangles in brain neurons.

The team decided to test how well their methods found O-GlcNAc proteins and started using tissue from either healthy or diseased mouse brains. The healthy tissue contained 274 different proteins marked with O-GlcNAc. However, many of these proteins had more than one sugar molecule, as the team discovered a total of 458 binding sites on those 274 proteins. This was three times the number of sites than any previous study had discovered, which allowed the team to find similarities between the 106 O-GlcNAc sites that were already identified in other studies, but also on the remaining 168 O-GlcNAc protein sites that had so far not been identified.

The team observed that these proteins had various functions, including forming part of a cell’s structure, in nerve growth or other nerve-related functions, like learning and memory. The team classified the 168 newly-identified proteins based on what they looked like and by what function they thought they would likely be involved in, i.e. cell signaling, regulating gene expression or structuring cells, before examining the proteins they found in the Alzheimer diseased mouse brains. They discovered that the diseased mouse brains contained about a third less O-GlcNAc-marked proteins, which supports earlier studies that indicated damaged O-GlcNAc regulation in the brains of people with Alzheimer’s.

Interestingly, the researchers discovered that over 98% of the O-GlcNAc proteins also had sites that would accept a phosphate, which indicates that these proteins are also controlled by the most common regulatory system in cells, the phosphate system.

The researchers found that approximately one quarter of the O-GlcNAc sites were in sufficiently close proximity to the phosphate sites in order to interfere with the switch, which indicates cross-communication between the two types of regulation. Whilst the phosphate is smaller than O-GlcNAc and has a strong negative electrical charge, the sugar is neutral yet bulkier. These characteristics could have different impacts on the protein’s structure, and furthermore, the range of potential biological outcomes could also be significantly higher due to the complexity of both switching systems.

Whilst the majority of the proteins that are known to be under O-GlcNAc control mostly live within the cells, the team also discovered that six proteins had to be controlled by O-GlcNAc outside a cell, depending on where their O-GlcNAc site was situated on the protein.

The team currently plans to examine both regulatory systems and Smith concludes:

“It’s 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.”

Written By Petra Rattue