Biology subjects

Peralta Martinez, R.

Publications and source records attributed to Peralta Martinez, R..

3 recordsLinked to original sources

bioRxiv2025

EXPERIMENTAL KINETIC MECHANISM OF P53 CONDENSATION-AMYLOID AGGREGATION

The tumor suppressor p53 modulates the transcription of a variety of genes constituting a protective barrier against anomalous cellular proliferation. High frequency "hot-spot" mutations result in loss-of-function by the formation of amyloid-like aggregates that correlate with cancerous progression. We show that full-length p53 undergoes spontaneous homotypic condensation at sub-micromolar concentrations and in the absence of crowders, to yield dynamic coacervates that are stoichiometrically dissolved by DNA. These coacervates fuse and evolve into hydrogel-like clusters with strong thioflavin-T binding capacity, which further evolve into fibrillar species with a clearcut branching growth pattern. The amyloid-like coacervates can be rescued by the HPV master regulator E2 protein to yield large regular droplets. Furthermore, we kinetically dissected an overall condensation mechanism which consists of a nucleation-growth process by sequential addition of p53 tetramers, leading to discretely-sized and monodisperse early condensates followed by coalescence into bead-like coacervates that slowly evolve to the fibrillar species. Our results suggest strong similarities to condensation-to-amyloid transitions observed in neurological aggregopathies. Mechanistic insights uncover novel key early and intermediate stages of condensation that can be targeted for p53 rescuing drug discovery. SIGNIFICANT STATEMENTKnown as "the guardian of the genome", the tumor suppressor protein p53 becomes activated by injuries to the DNA genome, and determines whether the cell must undergo self-destruction to avoid cancerous proliferation. P53 is in fact inactivated by mutations in over 50% of all cancers, and restoring its function is recognized as a therapeutic cancer target. A recent biochemical revolution in cell physiology and pathology are liquid entities known as biomolecular condensates. We show that p53 form condensates en route to pathological forms in a surprisingly similar manner to neurological amyloid diseases such as Alzheime[r]s and Parkinso[n]s. We uncover the sequence of steps in the reaction, exposing flanks for a novel drug development platform based on the condensates paradigm.

biophysics↗

bioRxiv2024

Protein-RNA condensation kinetics via filamentous nanoclusters

Protein-RNA phase separation is at the center of membraneless biomolecular condensates governing cell physiology and pathology. Using an archetypical viral protein-RNA condensation model, we determined the sequence of events that starts with sub-second formation of a protomer with two RNAs per protein dimer. Association of additional RNA molecules to weaker secondary binding sites in this protomer kickstarts crystallization-like assembly of a molecular condensate. Primary nucleation is faster than the sum of secondary nucleation and growth, which is a multistep process. Protein-RNA nuclei grow over hundreds of seconds into filaments and subsequently into nanoclusters with circa 600 nm diameter. Cryoelectron microscopy reveals an internal structure formed by incoming layers of protein-RNA filaments made of ribonucleoprotein oligomers, reminiscent of genome packing of a nucleocapsid. These nanoclusters progress to liquid condensate droplets that undergo further partial coalescence to yield typical hydrogel-like protein-RNA coacervates that may represent the scaffold of large viral factory condensates in infected cells. Our integrated experimental kinetic investigation exposes rate limiting steps and structures along a key biological multistep pathway present across life kingdoms.

biophysics↗

bioRxiv2022

The guardian of the genome meets a viral master gene regulator at a biomolecular condensate

As guardian of the genome, p53 exerts its tumor suppressor activity by modulating the expression of several hundreds of genes and by interacting with a large number of proteins. However, p53 can also repress viral replication and it is targeted by a variety of viral proteins to allow viral replication to proceed. p53 can repress human papillomavirus replication by binding to the viral E2 master gene regulator. Here we show how full-length p53 can spontaneously form phase separated liquid-like droplets that evolve to amyloid-like aggregates in a time-dependent manner, highlighting the fact that homotypic condensation is on the path to aggregation as observed in several protein aggregopathies. The DNA binding domain of HPV E2 (E2C) triggers heterotypic liquid-liquid phase separation with p53 with a precise 1 p53 : 2 E2C stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Moreover, E2C is able to slowly reshape time-evolved p53 aggregates into regular heterotypic liquid droplets. Using in situ sub-cellular fractionation, we show that E2 and wild-type p53 co-localize to the nucleus with a grainy pattern, and E2 can re-localize p53 into chromatin associated foci, a function independent of the DNA binding capacity of p53. A small DNA duplex containing the specific binding site for p53 deforms and dissolves both homotypic and heterotypic condensates at a 1 p53 : 1 DNA stoichiometry, whereas a [~]1000 base pair DNA fragment instead reshaped the condensates into distinct amorphous condensates containing p53, E2C and DNA, reminiscent of what we observe bound to chromatin. We conclude that p53 is a scaffold for liquid-liquid phase separation in line with its structural and functional features, in particular as a hub that binds multiple cellular protein partners as well as nucleic acids. Moreover, the capacity of E2C to rescue p53 from the amyloid aggregation route impacts on p53-rescuing drugs cancers where p53 mutation leads to loss of function.

biophysics↗