Charged sequence motifs increase affinity towards liquid-liquid phase separation

Protein phase separation has been shown to be a major governing factor in multiple cellular processes, especially ones concerning RNA and RNA-binding proteins. Despite many key observations, the exact structural characteristics of proteins involved in the process are still not fully deciphered. In this work we show that proteins harbouring sequences with specific regions of charged residues are significantly associated with phase separation phenomena. In particular, regions with repetitive arrays of alternating charges (termed charged residue repeats, CRRs) show the strongest association, whereas segments with generally high charge density (charge-dense regions, CDRs) and single alpha-helices (SAHs) show also detectable but weaker connections.It is known to contribute to the formation of membrane-less organelles (MLOs) and to an extent the aggregation of proteins. The causes and consequences of phase separation has been a rigorously researched topic in the last few years, as the condensation of specific phase-separating proteins is known to promote several diseases.In this work we carried out a computational analysis to examine the presence of repetitive segments with high charge density in proteins prone to phase separation. Free resources such as the Charged Single α-Helix (CSAH) web server and the PhaSepDB online database were used to examine possible links between the charged side-chain content of protein sequences and their partition into membrane-less condensates. Furthermore, we carried out the development of a novel algorithm aimed to detect a larger variety of charged protein segments, in order to examine their relationship to the phenomenon. Fisher’s exact test of independence was implemented on several generated data sets to confirm correlation between charged residue repeats (CRRs) and charge-dense regions (CDRs) within human protein sequences and their affinity for phase separation.

Electrostatic Interaction Between NS1 and Negatively Charged Lipids Contributes to Flavivirus Replication Organelles Formation

Flavivirus replication occurs in membranous replication compartments, also known as replication organelles (ROs) derived from the host ER membrane. Our previous study showed that the non-structural (NS) protein 1 (NS1) is the essential factor for RO creation by hydrophobic insertion into the ER membrane. Here, we found that the association of NS1 with the membrane can be facilitated by the electrostatic interaction between NS1 and negatively charged lipids. NS1 binds to a series of negatively charged lipids, including PI4P, and a positively charged residue, R31, located on the membrane-binding face of NS1, plays important roles in this interaction. The NS1 R31E mutation significantly impairs NS1 association with negatively charged membrane and its ER remodeling ability in the cells. To interfere with the electrostatic interaction between NS1 and negatively charged lipids, intracellular phosphatidylinositol phosphates (PIPs) level was downregulated by the overexpression of Sac1 or treatment with PI3K and PI4K inhibitors to attenuate flavivirus replication. Our findings emphasize the importance of electrostatic interaction between NS1 and negatively charged lipids in flavivirus RO formation.

N-Terminal Regions of Prion Protein: Functions and Roles in Prion Diseases

The normal cellular isoform of prion protein, designated PrPC, is constitutively converted to the abnormally folded, amyloidogenic isoform, PrPSc, in prion diseases, which include Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. PrPC is a membrane glycoprotein consisting of the non-structural N-terminal domain and the globular C-terminal domain. During conversion of PrPC to PrPSc, its 2/3 C-terminal region undergoes marked structural changes, forming a protease-resistant structure. In contrast, the N-terminal region remains protease-sensitive in PrPSc. Reverse genetic studies using reconstituted PrPC-knockout mice with various mutant PrP molecules have revealed that the N-terminal domain has an important role in the normal function of PrPC and the conversion of PrPC to PrPSc. The N-terminal domain includes various characteristic regions, such as the positively charged residue-rich polybasic region, the octapeptide repeat (OR) region consisting of five repeats of an octapeptide sequence, and the post-OR region with another positively charged residue-rich polybasic region followed by a stretch of hydrophobic residues. We discuss the normal functions of PrPC, the conversion of PrPC to PrPSc, and the neurotoxicity of PrPSc by focusing on the roles of the N-terminal regions in these topics.

Computational Investigations of the Transmembrane Italian-Mutant (E22K) 3A\(\beta_{11 - 40}\) in Aqueous Solution

The Amyloid beta (Aβ) oligomers are characterized as critical cytotoxic materials in Alzheimer’s disease (AD) pathogenesis. Structural details of transmembrane oligomers are inevitably necessary to design/search potential inhibitor due to treat AD. However, the experimental detections for structural modify of low-order Aβ oligomers are precluded due to the extremely dynamic fluctuation of the oligomers. In this project, the transmembrane Italian-mutant (E22K) 3Aβ11-40 (tmE22K 3Aβ11-40) was extensively investigated upon the temperature replica exchange molecular dynamics (REMD) simulations. The structural changes of the trimer when replacing the negative charged residue E22 by a positively charged residue K were monitored over simulation intervals. The oligomer size was turned to be larger and the increase of β-content was recorded. The momentous gain of intermolecular contacts with DPPC molecules implies that tmE22K 3Aβ11-40 easier self-inserts into the membrane than the WT one. Furthermore, the tighter interaction between constituting monomers was indicated implying that the E22K mutation probably enhances the Aβ fibril formation. The results are in good agreement with experiments that E22K amyloid is self-aggregate faster than the WT form. Details information of tmE22K trimer structure and kinetics probably yield the understanding of AD mechanism.