Research: Protein droplets may cause many types of genetic diseases

Most proteins are found in separate protein-rich droplets called "cellular condensates" in cells.

These proteins carry sequence characteristics that serve as address labels, informing the protein which condensates to move into.

Proteins may wind up in the wrong condensate if the labels are messed up.

According to a multinational team of clinical medicine and fundamental biology researchers, this could be the source of many unresolved disorders.

The findings were published in the journal Nature.

Patients with BPTA syndrome have characteristically malformed limbs featuring short fingers and additional toes, missing tibia bones in their legs and reduced brain size.

As the researchers found out, BPTAS is caused by a special genetic change that causes an essential protein to migrate to the nucleolus, a large proteinaceous droplet in the cell nucleus.

As a result, the function of the nucleolar condensate is inhibited and developmental disease develops.

In collaboration with scientists at the Max Planck Institute for Molecular Genetics (MPIMG) in Berlin, the University Hospital Schleswig-Holstein (UKSH), and contributors from all around the world, the team is pushing open a door to new diagnoses that could lead to the elucidation of numerous other diseases as well as possible future therapies.

Affected individuals have complex and striking malformations of the limbs, face, and nervous and bone systems, only partially described by the already-long disease name "brachyphalangy-polydactyly-tibial aplasia/hypoplasia syndrome" (BPTAS).

This protein has the task of organizing the genetic material in the cell nucleus and facilitates the interaction of other molecules with the DNA, for example, to read genes.

In mice, a complete loss of the gene on both chromosomes is catastrophic and leads to the death of the embryo.

In some patients with only one copy mutated, however, the cells are using the intact copy on the other chromosome, resulting only in mild neurodevelopmental delay.

But the newly discovered cases did not fit this scheme.

A closer look revealed that different mutations of HMGB1 have different consequences.

The sequencing data showed that in the affected individuals with severe malformations, the reading frame for the final third of the HMGB1 gene is shifted.

After translation to protein, the corresponding region is now no longer equipped with negative but with positively charged amino acid building blocks.

This can happen if a number of genetic letters not divisible by three are missing in the sequence because exactly three consecutive letters always code for one building block of the protein.

However, the tail part of the protein does not have a defined structure. Instead, this section hangs out of the molecule like a loose rubber band.

The purposes of such protein tails (also called "intrinsically disordered regions") are difficult to study because they often become effective only in conjunction with other molecules. So how might their mutation lead to the observed disease?

To answer this question, the medical researchers approached biochemists Denes Hnisz and Henri Niskanen at the MPIMG, who work with cellular condensates that control important genes.

These droplet-like structures behave much like the oil and vinegar droplets in a salad dressing. Composed of a large number of different molecules, they are separated from their surroundings and can undergo dynamic changes.

The nucleolus within the cell nucleus is also a condensate, which appears as a diffuse dark speck under the microscope.

This is where many proteins with positively charged tails like to linger.

Many of these provide the machinery required for protein synthesis, making this condensate essential for cellular functions.

The mutant protein HMGB1 with its positively charged molecular tail is attracted to the nucleolus as well, as the team observed from experiments with isolated protein and with cell cultures.

But since the mutated protein region has also gained an oily, sticky part, it tends to clump.

The nucleolus loses its fluid-like properties and increasingly solidifies, which Niskanen was able to observe under the microscope.

This impaired the vital functions of the cells - with the mutated protein, more cells in a culture died compared to a culture of cells without the mutation.

The research team then searched databases of genomic data from thousands of individuals looking for similar incidents.

In fact, the scientists were able to identify more than six hundred similar mutations in 66 proteins, in which the reading frame had been shifted by a mutation in the protein tail, making it both more positively charged and more "greasy". Of the mutations, 101 had previously been linked to several different disorders.

For a cell culture assay, the team selected 13 mutant genes. In 12 out of 13 cases, the mutant proteins had a preference to localize into the nucleolus. About half of the tested proteins impaired the function of the nucleolus, resembling the disease mechanism of BPTA syndrome.

But tumour diseases are also predominantly genetically determined, adds Hnisz: "Cellular condensates and the associated phase separation are a fundamental mechanism of the cell that also plays a role in cancer. The chances of developing targeted therapies for this are much better."