Artificial organelles created to control cellular behavior
New form of synthetic biology for controlling cellular behavior uses intrinsically disordered proteins

Date:
August 4, 2020
Source:
Duke University
Summary:
Biomedical engineers have demonstrated a method for controlling
the phase separation of an emerging class of proteins to create
artificial membrane-less organelles within human cells. The advance,
similar to controlling how vinegar forms droplets within oil,
creates opportunities for engineering synthetic structures to
modulate existing cell functions or create entirely new behaviors
within cells.



FULL STORY ========================================================================== Biomedical engineers at Duke University have demonstrated a method
for controlling the phase separation of an emerging class of proteins
to create artificial membrane-less organelles within human cells. The
advance, similar to controlling how vinegar forms droplets within oil,
creates opportunities for engineering synthetic structures to modulate
existing cell functions or create entirely new behaviors within cells.


==========================================================================
The results appear online on August 3 in the journal Nature Chemistry.

Proteins function by folding into specific 3-D shapes that interact
with different biomolecular structures. Researchers previously believed
that proteins needed these fixed shapes to function. But in the last two decades, a large new class of intrinsically disordered proteins (IDPs)
have been discovered that have large regions that are "floppy" -- that is,
they do not fold into a defined 3-D shape. It is now understood these
regions play an important, previously unrecognized role in controlling
various cellular functions.

IDPs are also useful for biomedical applications because they can undergo
phase transitions -- changing from a liquid to a gel, for example, or
from a soluble to an insoluble state, and back again -- in response to environmental triggers, like changes in temperature. These features also dictate their phase behavior in cellular environments and are controlled
by adjusting characteristics of the IDPs such as their molecular weight
or the sequence in which the amino acids are linked together.

"Although there are many natural IDPs that show phase behavior in cells,
they come in many different flavors, and it has been difficult to
discern the rules that govern this behavior," said Ashutosh Chilkoti,
the Alan L. Kaganov Distinguished Professor of Biomedical Engineering
at Duke. "This paper provides very simple engineering principles to
program this behavior within a cell." "Others in the field have taken
a top-down approach where they'll make a change to a natural IDP and
see how its behavior changes within a cell," said Michael Dzuricky, a
research scientist working in the Chilkoti laboratory and first author
of the study. "We're taking the opposite approach and building our own artificial IDPs from simple thermodynamic principles. This enables us and others to precisely tune a single property -- the shape of the IDPs phase diagram -- to better understand how this parameter affects biological
behavior" In the new paper, the researchers begin by looking to nature
for examples of IDPs that come together to form "biomolecular condensates" within cells. These weakly-held-together structures allow cells to create compartments without also building a membrane to encapsulate it. Using one
such IDP from the common fruit fly as a basis, the researchers draw from
their extensive history of working with IDPs to engineer a molecularly
simpler artificial version that retains the same behavior.



==========================================================================
This simpler version allowed the researchers to make precise changes
to the molecular weight of the IDP and amino acids of the IDPs. The
researchers show that, depending on how these two variables are
tweaked, the IDPs come together to form these compartments at different temperatures in a test tube. And by consistently trying various tweaks
and temperatures, the researchers gained a solid understanding of which
design parameters are most important to control the IDP's behavior.

A test tube, however, is not the same as a living cell, so the researchers
then went one step further to demonstrate how their engineered IDPs
behave within E.

coli. As predicted, their artificial IDPs grouped together to form a tiny droplet within the cell's cytoplasm. And because the IDP's behavior was
now so well understood, the researchers showed they could predictably
control how they coalesced using their test tube principles as a guide.

"We were able to change temperatures in cells to develop a complete
description of their phase behavior, which mirrored our test tube
predictions," said Dzuricky. "At this point, we were able to design
different artificial IDP systems where the droplets that are formed
have different material properties." Put another way, because the
researchers understood how to manipulate the size and composition of
the IDPs to respond to temperature, they could program the IDPs to form droplets or compartments of varying densities within cells. To show how
this ability might be useful to biomedical engineers, the researchers
then used their newfound knowledge, as nature often does, to create an organelle that performs a specific function within a cell.

The researchers showed that they could use the IDPs to encapsulate an
enzyme to control its activity level. By varying the molecular weight of
the IDPs, the IDPs hold on the enzyme either increased or decreased, which
in turn affected how much it could interact with the rest of the cell.



==========================================================================
To demonstrate this ability, the researchers chose an enzyme used by
E. coli to convert lactose into usable sugars. However, in this case,
the researchers tracked this enzyme's activity with a fluorescent reporter
in real-time to determine how the engineered IDP organelle was affecting
enzyme activity.

In the future, the researchers believe they could use their new IDP
organelles to control the activity levels of biomolecules important to
disease states. Or to learn how natural IDPs fill similar cellular roles
and understand how and why they sometimes malfunction.

"This is the first time anybody has been able to precisely define how
the protein sequence controls phase separation behavior inside cells,"
said Dzuricky. "We used an artificial system, but we think that the
same rules apply to natural IDPs and are excited to begin testing this
theory." "We can also now start to program this type of phase behavior
with any protein in a cell by fusing them to these artificial IDPs,"
said Chilkoti. "We hope that these artificial IDPs will provide new
tool for synthetic biology to control cell behavior." This research
was supported by the National Institutes of Health (R35GM127042) and
the National Science Foundation (DMR-17-29671, CHE-1709735).


========================================================================== Story Source: Materials provided by Duke_University. Original written
by Ken Kingery. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Michael Dzuricky, Bradley A. Rogers, Abdulla Shahid, Paul S. Cremer,
Ashutosh Chilkoti. De novo engineering of intracellular condensates
using artificial disordered proteins. Nature Chemistry, 2020; DOI:
10.1038/ s41557-020-0511-7 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/08/200804122214.htm

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