Scientific Frontline: Finding Order in the Disorder: New Insights into Seemingly Structureless Regions of Proteins

Wednesday, October 18, 2023

Finding Order in the Disorder: New Insights into Seemingly Structureless Regions of Proteins

Protein condensates (shown here in a microscope image) are critical to the process of gene expression in cells, and condensate formation depends on proteins’ intrinsically disordered regions.
Image Credit: Amy Strom, Princeton University

Howard Hughes Medical Institute Investigators Cigall Kadoch and Clifford Brangwynne teamed up to challenge long-held beliefs in the scientific community about how – or even if – structureless, unorganized regions of proteins play specific roles in causing or preventing disease pathogenesis. Their work was published earlier this month in the journal Cell.

To understand the significance of the duo’s findings, it helps to first dive deep into the ways in which cells alter genomic structure through a process known as chromatin remodeling. About two meters – or six and a half feet – of DNA are packed inside each cell’s nucleus, which measures no larger than a pinhead. To fit in that space, DNA winds around proteins, forming a compact structure called chromatin. At the Dana-Farber Cancer Institute, Kadoch and her lab have spent years studying chromatin remodeling complexes – molecular machines made up of multiple proteins that change the physical structure of chromatin and, thus, suppress or enable the activity of genes in a programmatic manner.

In recent years, her lab’s focus has centered on a family of complexes known as mammalian SWI/SNF chromatin remodeling complexes – commonly referred to as BAF complexes – which have garnered significant attention due to their disruption and involvement in human disease. BAF complexes are one of the most frequently mutated cellular entities in human cancer, second only to TP53, a well-studied tumor suppressor gene. Studies have shown that approximately 20 percent of human cancers bear BAF complex mutations and that such disruptions are also among the most common in neurodevelopmental disorders (NDDs) such as autism and intellectual disability.

This microscope image shows BAF complex condensates in human cells.
Image Credit: Kadoch and Brangwynne labs, Dana-Farber Cancer Institute and Princeton University

Within these BAF complexes, most mutations occur in one of two genes — ARID1A or ARID1B — and more than half the time, the mutations are found in regions of these proteins known as intrinsically disordered regions (IDRs). Scientists have long believed that these sticky, seemingly structureless regions are nonspecific, and that they basically function to grip onto other proteins no matter the amino acid composition of these regions. These regions are, however, known to play a role in forming biomolecular condensates, membrane-less regions of high local protein concentration formed via the process of phase separation, whereby biological molecules in cells undergo transitions, similar to how water vapor transitions to a liquid through condensation.

But, when it comes to chromatin remodelers, it’s been unclear what role – if any – IDRs might play in their overall function; the prevailing assumption has been that IDRs carry out nonspecific functions as if they’re merely stickers that adhere to and bind proteins.

Kadoch and her team wanted more than assumptions – after all, many in the scientific community and in the pharmaceutical industry see chromatin remodelers as a potential avenue for revolutionary new treatments for cancers and other diseases. Given that more than half of the most commonly occurring mutations in ARID1A and ARID1B found in cancer and neurodevelopmental disorders, respectively, take place in IDRs, Kadoch and her team knew they had to challenge longstanding dogma and define the impact of IDRs on BAF complex function.

“If we could understand the functional contributions of these intrinsically disordered sequences, we would have a better understanding mechanistically of what’s driving disease,” says Kadoch. “Most importantly, we might have a new toehold for being able to explore therapeutic opportunities for a very large number of people, including those with cancer, autism, immune conditions, and other disorders driven by lesions in these genes.”

A closer look at IDRs

To learn more about these enigmatic IDRs, the Kadoch lab joined forces with Brangwynne and members of his HHMI lab at Princeton University who are experts in developing optogenetic platforms and other biophysical tools to study and understand IDRs.

Together, the Kadoch and Brangwynne labs challenged assumptions about IDR composition by asking two main questions: Would changes to IDR composition affect BAF condensate formation, and would such changes affect the ability of BAF complexes to engage with the right proteins in the nucleus, target to the proper sites along DNA, and activate certain genes?

To find the answers, researchers from both labs worked together to swap IDRs found in ARID1A proteins with IDRs from other well-known phase separation-prone proteins. As suspected, the group’s swap with the new ARID1A mutant variants made no difference in the process of condensate formation, confirming that “similar phase-separation-prone sequences would do” when it comes to yielding condensates, Kadoch says.

But, when the groups tested how the mutant ARID1A variants affected genomic targeting and protein interactions of BAF complexes, the results painted a very different picture of IDRs, one that now changes existing paradigms in the field. The chromatin remodelers slipped out of position, losing their ability to localize to their appropriate sites along chromatin, and hence, failed to establish the appropriate gene expression pattern.

“These chromatin remodelers have to be able to go to the right sites at the right time, to activate the right genes, or they can cause disease,” Kadoch says. “We found that BAF complexes cannot tether the right repertoire of proteins in the nuclei of cells with any protein sequence capable of phase separation and condensation, rather, they require the specific sequence – in this case, that of ARID1A/B subunits – to do that. This finding shows that IDRs have inherent specificity required for their evolutionarily adapted functions.”

Mapping the "grammar"

To better understand this specificity, Kadoch and Brangwynne enlisted the expertise of Rohit Pappu, a biophysicist at Washington University in St. Louis. Pappu and his team helped map the code – or “grammar” – of the IDRs to confirm what Kadoch and Brangwynne suspected: ARID1A/B proteins have unique amino acid sequences that play a critical role in the protein-protein interaction network. “When we mess with these specific sequences, not just those that enable condensate formation, we prevent BAF complexes from going where they’re supposed to go on the genome,” Kadoch says. “The result is dysregulation of chromatin accessibility and ultimately, gene expression.”

“In the biomolecular condensate field, we are focused on understanding molecular specificity and functional consequences,” Brangwynne says. “We’re really excited about this paper because it is a particularly clear example of how IDR sequences can encode for both condensate formation and a very specific set of partner interactions – and both are critical for proper chromatin remodeling function.”

The group’s findings could change conventional thinking about chromatin remodeling and also have a cascading effect that leads scientists to ask new questions about amino acid sequences in proteins with IDRs all over the cell, in a wide range of different cell types.

“This presents an important set of biophysical and biological paradigms that are useful to the entire field of cell biology and basically all of life sciences,” Kadoch says. “What this teaches the field is that there’s a very specific grammar sequence [encoded in IDRs] that is a biologic intention, achieved through evolution, that accommodates very specific binding interactions within biomolecular condensates. This is the first time this has been demonstrated and we are excited by its broad-spanning applicability.”

Kadoch and Brangwynne feel their work drives home the importance of interdisciplinary collaboration in science.

“Diversity in science, diversity in thinking, diversity in expertise and skillsets are absolutely critical to our ability, as researchers, to uncover new findings about some of the most fundamental biochemical and biophysics principles governing life,” Kadoch says. “I think this work showcases the importance of collaborations in modern science.”

Published in journal: Cell

Research Material: