Biology subjects

Perdikari, T. M.

Publications and source records attributed to Perdikari, T. M..

6 recordsLinked to original sources

bioRxiv2022

Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation

1,6-hexanediol disrupts many phase-separated condensates in cells and in test tubes. In this study, we use a combination of microscopy, nuclear magnetic resonance (NMR) spectroscopy, molecular simulation, and biochemical assays to probe how alkanediols suppress phase separation and why certain isomers are more effective. Alkanediols of different lengths and configurations are all capable of disrupting phase separation of the RNA-binding protein Fused in Sarcoma (FUS), though potency varies depending on both geometry and hydrophobicity, which we measure directly. Alkanediols induce a shared pattern of changes to the protein chemical environment though to differing extents. Consistent with the view that alkanediols disrupt phase separation driven by hydrophobic groups, they decrease the thermal stability of a model globular protein. Conversely, 1,6-hexanediol does not disrupt charge-mediated phase separation, such as FUS RGG-RNA and poly-lysine/poly-aspartic acid condensates. All-atom simulations show that hydroxyl groups in alkanediols mediate interaction with protein backbone and polar amino acid side chains, while the aliphatic chain allows contact with hydrophobic and aromatic residues, providing a molecular picture of how amphiphilic interactions disrupt FUS phase separation.

biochemistry↗

bioRxiv2021

The oncogenic transcription factor FUS-CHOP can undergo nuclear liquid-liquid phase separation

Myxoid liposarcoma is caused by a chromosomal translocation resulting in a fusion protein comprised of the N-terminus of FUS (fused in sarcoma) and the full-length transcription factor CHOP (CCAAT/Enhancer Binding Protein Homologous Protein). FUS functions in RNA metabolism and CHOP is a stress-induced transcription factor. The FUS-CHOP fusion protein causes unique gene expression and oncogenic transformation. The FUS segment is required for oncogenic transformation, but the mechanism of FUS-CHOP-induced transcriptional activation is unknown. Recently, some transcription factors and super enhancers were proposed to undergo liquid-liquid phase separation and form membraneless compartments that recruit transcription machinery to gene promoters. Since phase separation of FUS depends on its N-terminus, transcriptional activation by FUS-CHOP could result from the N-terminus driving nuclear phase transitions. Here, we characterized FUS-CHOP in cells and in vitro, and observed novel phase-separating properties relative to unmodified CHOP. Our data indicate FUS-CHOP forms phase-separated condensates at super enhancer transcriptional sites. We provide strong evidence that the FUS-CHOP phase transition is a novel oncogenic mechanism and potential therapeutic target for treatment of myxoid liposarcoma.

cell biology↗

bioRxiv2021

An enhancer sequence in the intrinsically disordered region of the essential cell division protein FtsZ promotes conformation-guided substrate processing by ClpXP in Escherichia coli

The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation by conformationally-guided interactions.

microbiology↗

bioRxiv2020

A predictive coarse-grained model for position-specific effects of post-translational modifications on disordered protein phase separation

Biomolecules undergo liquid-liquid phase separation (LLPS) resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed charge atomistic forcefields, we parameterize the size and atomistic hydropathy of the coarse-grained modified amino acid beads, and hence the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the proteins single chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of FUS and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution. Statement of SignificancePost-translational modifications are important regulators of liquid-liquid phase separation (LLPS) which drives the formation of biomolecular condensates. Theoretical methods can be used to characterize the biophysical properties of intrinsically disordered proteins (IDPs). Our recent framework for molecular simulations using a C-centered coarse-grained model can predict the effect of various perturbations such as mutations (Dignon et al. PloS Comput. Biol, 2018) and temperature (Dignon et al, ACS Cent. Sci., 2019) on LLPS. Here, we expand this framework to incorporate modified residues like phosphothreonine, phosphoserine and acetylysine. This model will prove useful for simulating the phase separation of post-translationally modified IDPs and predicting how position-specific modifications can control phase behavior across the large family of proteins known to be phosphorylated and acetylated.

biophysics↗

bioRxiv2020

SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.

biochemistry↗

bioRxiv2020

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration

mRNA transport in neurons is a ubiquitous process but has been often overlooked as a contributor to disease. Mutations of transport granule protein hnRNPA2 cause hereditary proteinopathy of neurons, myocytes, and bone. Here, we examine transport granule component specificity, assembly/disassembly, and the link to neurodegeneration. hnRNPA2 transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet they each partition specifically into hnRNPA2 liquid phases. hnRNPA2 tyrosine phosphorylation dissociates granule interactions by reducing hnRNPA2 phase separation and preventing partitioning of hnRNPF and ch-TOG; tyrosine phosphorylation also decreases aggregation of hnRNPA2 disease mutants. A C. elegans model of hnRNPA2 D290V-associated neurodegeneration exhibits TDP-43 ortholog-dependent glutamatergic neurodegeneration. Expression of the tyrosine kinase that phosphorylates hnRNPA2 reduces glutamatergic neurodegeneration. The evidence for specific partitioning of granule components as well as disruption of these interactions and reduction of neurodegeneration by tyrosine phosphorylation suggest transport granule biology has a role in the pathogenesis of neurodegeneration.

biochemistry↗