Mechanisms Regulating Phase Separation And Aggregation Of Proteins Associated With Neurodegenerative Diseases

Neurodegenerative diseases are a group of diseases in which the gradual loss of neuronal cell structure or function or even death leads to dysfunction,which seriously endangers human health.Amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD)are two common neurodegenerative diseases with similar pathological features,namely the presence of aggregates of proteins in the brain and spinal cord of patients.Many membraneless organelles,such as nucleoli and stress granules,exist on cells and regulate many important biological processes.Many of the proteins associated with ALS/FTD were found in the membraneless organelles of neurons.The formation of membraneless organelles is driven by liquid-liquid phase separation(LLPS).Droplets formed by LLPS can be converted into amyloid fibrils by an abnormal irreversible phase transition.The amyloid fibrils are the aggregates observed in neurons of ALS/FTD patients.Multiple proteins(including FUS and TIA1)are present in neurons of ALS/FTD patients,and the low-complexity domains(LCDs)of these proteins tend to promote aggregation and phase separation.However,the relationship between FUS and TIA1 aggregation and LLPS,as well as the regulatory mechanisms,remains to be investigated.In this dissertation,the regulatory mechanism of the phase separation and aggregation of the proteins FUS and TIA1 was investigated as follows.1.The identification of the amyloid segments of FUS and the study of its effect on phase separation.It was recently found that FUS-LCD can form amyloid fibrils,and structural studies showed that a fragment of 57 residues constitutes the core of the FUS-LCD fibril structure.Based on this,we divided the FUS-LCD core fragment into four peptides with similar sequences,named R1-R4,to explore the shorter amyloid-aggregating fragment of FUS.By characterizing the ability of the peptides to aggregate,the morphological characteristics,the stability,and the secondary structure of the aggregates,we found that only R2 was able to form amyloid fibrils with a β-sheet structure.R2 fibrils have temperature reversibility.Further study revealed that R2 could induce the aggregation of FUS-LCD in phase separated droplets.We revealed an intermediate state of fibril growth inside of the droplet,which provides new insights into how functional phase separated FUS aggregates to form pathogenic proteins.2.Study of the regulatory mechanism of site-specific phosphorylation on FUS phase separation and aggregation.We first investigated the effect of Ser54 and Ser61 phosphorylation on the aggregation based on the R2 of FUS.The morphological observation of aggregation by atomic force microscopy and transmission electron microscopy revealed that Ser61 phosphorylation had a more significant effect on inhibiting the aggregation of R2.Toxicity experimental studies showed that Ser61 phosphorylation reduced cytotoxicity.Further,we obtained the full-length FUS protein by cell-free expression system and found that R2 could act as a seed to induce the aggregation of full-length protein FUS,However,Ser61 phosphorylation disrupted the seeding ability.Finally,the molecular mechanism by which Ser61 phosphorylation affects aggregation was investigated by molecular dynamics simulations and solid-state NMR spectroscopy.Ser61 phosphorylation disrupts the intramolecular and intermolecular interactions that maintain the parallel β-sheet structure of R2.Our results suggest that site-specific phosphorylation plays a key role in regulating the phase separation and aggregation of FUS.3.Characterization of liquid-liquid phase separation properties of TIA1.we initially observed phase separation of full-length TIA1(FL-TIA1)and TIA1-LCD proteins by confocal microscopy in vitro.Further,we found that high ionic strength,i.e.electrostatic interactions,could inhibit TIA1-LCD liquid-liquid phase separation.In the temperature range of 4-50°C,high temperature inhibited the liquid-liquid phase separation of TIA1-LCD.Further,we constructed eukaryotic plasmids of FL-TIA1 and TIA1-LCD and expressed the proteins intracellularly by transient transfection.Confocal microscopy,fluorescent bleaching recovery experiments observed that phase separation of FL-TIA1 and TIA1-LCD could occur intracellularly to form droplets with mobility.These results lay the foundation for further study of the regulatory mechanism of TIA1 phase separation.4.Regulation of TIA1 phase separation and aggregation by proline-related mutations.Proline inhibits the formation of amyloid-like aggregates,and recent studies have revealed that proline-rich regions of certain protein regulate the protein-liquid phase separation process.We found that proline-related mutations in LCD altered the droplet morphology and mobility of TIA1-LCD compared to wild-type TIA1-LCD by confocal microscopy,fluorescent bleaching recovery experiments.Mutation studies on FL-TIA1 revealed that proline-leucine(P-L)mutations accelerated TIA1 fibrilization.Further intracellular experiments on FL-TIA1 revealed that P-L mutations(including P352 L,P362L)phase separated droplet mobility was reduced and multiple proline mutations(11P-A,11 proline mutations to alanine)led directly to aggregation.In addition,all these proline mutations(P352L,P362 L,11P-A)delayed the clearance of intracellular stress granules.These results suggest that proline may be a key residue regulating TIA1 phase separation and aggregation.In summary,this study identified an FUS amyloid fibril fragment and used it as a basis to elucidate the mechanism of site-specific phosphorylation regulation of FUS proteins,as well as the mechanism of liquid-liquid phase separation and aggregation regulated by TIA1 proline-related mutations.The phosphorylation and mutation regulation mechanism,protein structure model and phase separation to aggregation transition mechanism can provide a basis for further research on related proteins.