Expanding the molecular grammar of polar residues and arginine in FUS prion-like domain phase separation and aggregation.

A molecular grammar governing low-complexity prion-like domains phase separation (PS) has been proposed based on mutagenesis experiments that identified tyrosine and arginine as primary drivers of phase separation via aromatic-aromatic and aromatic-arginine interactions. Here we show that additional residues make direct favorable contacts that contribute to phase separation, highlighting the need to account for these contributions in PS theories and models. We find that tyrosine and arginine make important contacts beyond only tyrosine-tyrosine and tyrosine-arginine, including arginine-arginine contacts. Among polar residues, glutamine in particular contributes to phase separation with sequence/position-specificity, making contacts with both tyrosine and arginine as well as other residues, both before phase separation and in condensed phases. For glycine, its flexibility, not its small solvation volume, favors phase separation by allowing favorable contacts between other residues and inhibits the liquid-to-solid (LST) transition. Polar residue types also make sequence-specific contributions to aggregation that go beyond simple rules, which for serine positions is linked to formation of an amyloid-core structure by the FUS low-complexity domain. Hence, here we propose a revised molecular grammar expanding the role of arginine and polar residues in prion-like domain protein phase separation and aggregation.

Structure and interactions of prion-like domains in transcription factor Efg1 phase separation.

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Efg1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered but have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase-separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans transcription factor interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.

Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation.

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Egf1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered though have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans TF interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.

A Spectrophotometric Turbidity Assay to Study Liquid-Liquid Phase Separation of UBQLN2 In Vitro.

Liquid-liquid phase separation (LLPS) is hypothesized to be the underlying mechanism for how membraneless organelles or biomolecular condensates form inside both prokaryotic and eukaryotic cells. Protein LLPS is a biophysical process during which proteins demix from homogeneous solution to form protein-dense droplets with liquid-like properties. Disruptions to LLPS, such as changes to material properties of condensates or physicochemical parameters for LLPS onset, are implicated in neurodegenerative diseases and cancer. Therefore, it is essential to determine the physicochemical parameters that promote protein LLPS. Here, we present our UV-Vis spectrophotometric turbidity assay to characterize the temperature and concentration dependence of LLPS for UBQLN2, a protein that undergoes LLPS via homotypic interactions in vitro and forms stress-induced condensates in cells. Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) and disrupt UBQLN2 LLPS. We present a detailed expression and purification protocol for a C-terminal construct of UBQLN2 and how we use microscopy to image UBQLN2 LLPS. We use our UV-Vis assay to construct temperature-concentration phase diagrams for wild-type and mutant UBQLN2 constructs to determine the effects of domain deletions and/or mutations on UBQLN2 phase separation.

Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation.

Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are low-complexity STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1-I and residues 555-570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2.

Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism.

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the "CTD code" that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS)26 of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4-5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.

Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery.

Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin-proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.

Spectroscopic studies on the interaction of carteolol hydrochloride and urea-induced bovine serum albumin.

The interaction of carteolol hydrochloride, to 0.2 mol L(-1) urea-induced bovine serum albumin in aqueous solution has been first investigated by fluorescence spectra and ultraviolet-visible (UV-vis) spectra at pH 7.40. The quenching mechanism, binding parameter and sites (n), the binding mode (ΔG, ΔH, and ΔS) as well as the binding distance (r) have been obtained according to the experimental results. We also use the synchronous fluorescence method to study the effect of CTL on the conformation change of urea-induced BSA.