Biology subjects

Loris, R.

Publications and source records attributed to Loris, R..

17 recordsLinked to original sources

bioRxiv2026

Multivalent ubiquitin recognition regulates multiple stages of Tau self-assembly

Liquid-liquid phase separation (LLPS) has emerged as an important intermediate in the pathological self-assembly of Tau, yet the molecular mechanisms that regulate this process remain incompletely understood. Although ubiquitin is a prominent component of pathological inclusions in tauopathies and other neurodegenerative diseases, its presence has largely been linked to targeting aberrant proteins for proteasomal or autophagic clearance. Here, we identify Tau as a ubiquitin-binding protein and demonstrate that multivalent, non-covalent ubiquitin recognition regulates multiple stages of Tau self-assembly. Biochemical, biophysical, structural, and computational analyses identified four ubiquitin-binding elements within the Tau microtubule-binding repeat region that engage the canonical hydrophobic patch of ubiquitin, providing a structural basis for multivalent recognition of mono- and poly-ubiquitin species. Functionally, mono- and poly-ubiquitin suppress Tau condensate formation while enhancing the liquid-like properties of condensates that persist. In contrast, mono-ubiquitin selectively enhances aggregation of the disease-linked Tau-P301L variant under conditions without detectable LLPS, demonstrating that ubiquitin exerts distinct effects depending on the molecular context and stage of Tau self-assembly. Together, these findings establish non-covalent ubiquitin recognition as a previously unrecognized mechanism regulating Tau phase behavior and aggregation. More broadly, they expand the role of ubiquitin beyond protein quality control, with ubiquitin architecture providing an additional layer of regulation over Tau condensate formation, dynamics, maturation, and aggregation.

biophysics↗

bioRxiv2026

Disease-linked mutations in hnRNPA2B1 accelerate condensate maturation and promote amyloid aggregation

Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is a multifunctional RNA-binding protein that undergoes liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles across different cellular contexts. However, dysregulated phase separation can drive the transition from functional condensates to pathological protein aggregates. Two mutations in the low-complexity domain (LCD) of hnRNPA2B1 (D302V and P310L) have been linked to neurodegenerative diseases. How these mutations alter the interplay between LLPS and aggregation remains poorly understood. Here, we show that these disease-linked mutations accelerate condensate maturation and promote amyloid-like aggregation of hnRNPA2B1, with fibrils emerging from condensate-like cores. To dissect the mechanistic link between phase separation, aggregation, and disease, we focused on a conserved 25-amino-acid region within the LCD harboring both mutation sites. Deletion of this region markedly impairs LLPS and abolishes aggregation, revealing its contribution to both processes. Isolated peptides derived from this region have an intrinsic propensity for amyloid-like fibril formation, which is enhanced by the mutations. Although unable to phase separate, these peptides remodel condensate morphology and nucleate aggregation of the LCD. Together, our results identify a sequence-encoded mechanism linking disease-associated mutations to altered condensate behavior and amyloid aggregation, providing molecular insights into how aberrant phase transitions may contribute to hnRNPA2B1-associated disease.

biochemistry↗

bioRxiv2026

Binding of long non-coding RNAs within the androgen receptor N-terminal tail modulates interdomain communication and liquid-liquid phase separation

The androgen receptor (AR) is a transcription factor whose overactivation is a primary driver of prostate cancer. Although AR interactions with several long non-coding RNAs (lncRNAs) have been implicated in castration-resistant prostate cancer, their underlying molecular mechanisms and functional consequences remain poorly understood. Here, we identify the N-terminal 37 residues of the intrinsically disordered AR N-terminal domain as the primary RNA-binding region that mediates selective interactions with the lncRNAs HOTAIR and SLNCR1. Residue-level mapping and mutational analysis define Y11, R13, and Q24 as key determinants of RNA recognition. This RNA-binding region partially overlaps with the F23QNLF27 motif, previously shown to mediate N/C interdomain communication with the ligand-binding domain through folding upon binding. We observe a partner-dependent binding mode in which this motif remains dynamically disordered upon RNA binding. LncRNA binding promotes phase separation of the N-terminal domain, indicating that lncRNAs upregulated in late-stage prostate cancer may lower the threshold for AR condensate formation and contribute to ligand-independent AR signaling. LncRNAs modulate communication between the N-terminal and ligand-binding domains within condensates, suggesting that lncRNA binding may tune hormone-dependent full-length AR signaling. These findings provide a mechanistic framework for AR-lncRNA regulation, laying the groundwork for future therapeutic strategies against advanced prostate cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/728402v1_ufig1.gif" ALT="Figure 1000"> View larger version (22K): org.highwire.dtl.DTLVardef@bee76dorg.highwire.dtl.DTLVardef@14bb6edorg.highwire.dtl.DTLVardef@1d693b5org.highwire.dtl.DTLVardef@7b70d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Ionic strength modulates structural disorder and protein oligomerization in the marginally disordered Phd transcription factor

Some proteins combine sequence features that are typical both for folded proteins and intrinsically disordered proteins (IDPs). The borderline properties of these so-called "marginal" IDPs render their conformational ensembles highly sensitive to the environmental changes, which may be important for their function. Here, we investigate the prokaryotic transcription factor Phd, which regulates the phd-doc toxin-antitoxin module through an allosteric mechanism involving disorder-order transition. Using an ensemble of biophysical techniques, we show that the protein is completely disordered at low ionic strength, whereas increasing salt concentration promotes its collapse into a partially ordered monomeric state, followed by the formation of a structured dimer. Using a thermodynamic model, we decipher the linkage between ionic strength, protein stability, oligomer state and degree of disorder. Via small-angle X-ray scattering we derive the structural ensemble of dimeric Phd, revealing a gradation of disorder as a function of salt. The sequence and biophysical properties of Phd position it at the boundary between macroscopically distinct conformational ensembles, representing a large pool of states capable of engaging in functional disorder-to-order interactions, enabling Phd to act as conformational rheostat. Together with previous crystallographic data, this charts the full spectrum of disorder-to-order states in the bacterial transcription factor and underscores the structural plasticity of IDPs with the marginal sequence properties. SIGNIFICANCEWhile globular proteins adopt stable three-dimensional structures, intrinsically disordered proteins (IDPs) remain flexible and dynamically sample partially ordered states. This behavior is largely determined by amino acid composition; however, some proteins lie at the borderline between order and disorder. Here, we focus on such a protein, the Phd transcription, and show how its conformation changes from completely disordered to fully ordered. These transitions are modulated by ionic strength and binding to macromolecules, including homodimerization. Using a thermodynamic model, we map the Phd conformational space, revealing a broad ensemble of states with varying degrees of disorder. The borderline amino acids properties enable Phd to function as a conformational rheostat, coupling functional interactions to the series of graded order-disorder transitions.

biophysics↗

bioRxiv2025

Inhibition of Gyrase via a conserved α-hairpin in Vibrio cholerae ParE2 and neutralization by ParD2

The ParE family of toxins is known to target Gyrase through an as yet unknown molecular mechanism. Here we show that Vibrio cholerae ParE2 (VcParE2) interacts with Gyrase in a DNA-dependent manner. The interaction site is located at the ParE-specific -hairpin and involves Trp25 as a key residue. The latter is poorly conserved within the ParE family, although full toxicity is only retained upon substitution with Tyr in the VcParE2 context. In vitro, the Trp25Ala mutation reduces the rate of Gyrase-mediated supercoiling, while the so-called cleavable complex remains stabilized. The C-terminal domain of the antitoxin VcParD2, which wraps its intrinsically disordered domain around VcParE2 without covering the Trp25-containing interaction site, binds to VcParE2 with high affinity, but only partially prevents ParE-mediated growth inhibition. Full inhibition of VcParE2 requires full length VcParD2 that leads to a complex where Trp25 is fully shielded from solvent.

biochemistry↗

bioRxiv2025

Expression and Purification of Full-Length hnRNPA2B1 for Biophysical Characterization of Liquid-Liquid Phase Separation

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is a multifunctional RNA-binding protein involved in RNA maturation and mRNA transport. It has recently been shown to undergo liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles. Moreover, dysregulation of LLPS is associated with the formation of pathogenic protein aggregates, in which hnRNPA2B1 is frequently found. Despite its biological and pathological relevance, studies on the full-length protein remain limited due to its intrinsically disordered, low-complexity domain, which renders hnRNPA2B1 highly aggregation-prone and difficult to purify. In this study, we report the successful expression and purification of full-length hnRNPA2B1 with high purity and minimal nucleic acid contamination. By optimizing buffer conditions, specifically ionic strength and pH, we maintain the protein in solution following cleavage of its solubility tag. Preliminary in vitro characterization under near-physiological conditions reveals that purified hnRNPA2B1 undergoes LLPS, forming dynamic liquid-like droplets that grow and mature into amorphous aggregates. Our approach provides a robust method for purifying hnRNPA2B1 suitable for LLPS and aggregation studies. This strategy may also be useful to purify other aggregation-prone, intrinsically disordered proteins.

biochemistry↗

bioRxiv2025

DNA-binding and dimerization of the SOG1 NAC domain are functionally linked with its ability to undergo liquid-liquid phase separation

Liquid-liquid phase separation is a key phenomenon in the regulation of transcription in eukaryotes leading to the formation of so-called membraneless organelles. While transcription factors take part in several types of membrane-less organelles, it remains unclear how specific DNA binding, multivalent interactions with DNA/RNA and condensation are interlinked. Here we show that the NAC domain of SOG1 (SOG1NAC), a transcription factor that is central to the DNA damage response in plants, can undergo liquid-liquid phase separation in vitro in the presence of both RNA or double stranded DNA. This behaviour, as well as the ability of SOG1NAC to bind DNA in a sequence-specific manner are dependent on its potential to form homodimers and the presence of a cluster of positive charges in its DNA binding site. Short double-stranded DNA fragments containing the sequence motif that is specifically recognized by SOG1NAC inhibit RNA-mediated phase separation, suggesting overlapping binding sites for DNA and RNA. This may reflect a complex interplay between DNA and RNA binding that could control the formation of condensates at transcription sites.

biochemistry↗

bioRxiv2022

Structural insights into Charcot-Marie-Tooth disease-linked mutations in human GDAP1

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its different subtypes are linked to mutations in dozens of different genes. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause two types of CMT, demyelinating CMT4A and axonal CMT2K. The GDAP1-linked CMT genotypes are mainly missense point mutations. Despite clinical profiling and in vivo studies on the mutations, the etiology of GDAP1-linked CMT is poorly understood. Here, we describe the biochemical and structural properties of the Finnish founding CMT2K mutation H123R as well as CMT2K-linked R120W, both of which are autosomal dominant mutations. The disease variant proteins retain close to normal structure and solution behaviour, but both present a large decrease in thermal stability. Using GDAP1 variant crystal structures, we identify a side chain interaction network between helices 3, 6, and 7, which is affected by CMT mutations, as well as a hinge in the long helix 6, which is linked to structural flexibility. Structural analysis of GDAP1 indicates that CMT may arise from disruption of specific intra- and intermolecular interaction networks, leading to alterations in GDAP1 structure and stability, and eventually, insufficient motor and sensory neuron function.

biochemistry↗

bioRxiv2021

High-affinity nanobodies as tools for structural and functional studies on mammalian Arc

Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro. This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo.

biochemistry↗

bioRxiv2021

Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was recently identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.

biophysics↗

bioRxiv2021

Vibrio cholerae ParE2 toxin modulates its operon transcription by stabilization of an antitoxin DNA ruler

The parDE2 operon of Vibrio cholerae encodes a type II TA system, which is one of three loci in the superintegron of small chromosome II that show modest similarity to the parDE operon of plasmid RK2. ParE2, like plasmid RK2-encoded ParE, inhibits DNA gyrase, an essential topoisomerase that is also the target of quinolone antibacterial agents. Mechanistic understanding on ParE2 toxin inhibition by direct interaction with its cognate antitoxin and transcriptional autoregulation of the TA system are currently lacking. ParD2, the ribbon-helix-helix (RHH) antitoxin, auto-represses the parDE2 promoter. This repression is enhanced by ParE2, which therefore functions as a transcriptional co-repressor. Here we present protein-DNA interaction studies and high-resolution X-ray structures of the ParD2:ParE2 complex and isolated ParD2 antitoxin, revealing the basis of toxin inhibition and autoregulation of the TA operon by conditional cooperativity. Native mass spectrometry, SAXS and MALS studies confirm the presence of different oligomerization states of ParD2 in solution and the role of the DNA-binding hexameric ParD26:ParE22 assembly in transcriptional repression.

microbiology↗

bioRxiv2021

Entropic pressure controls oligomerization of Vibrio cholerae ParD2 antitoxin

ParD2 is the antitoxin component of the parDE2 toxin-antitoxin module from Vibrio cholerae and consists of an ordered DNA binding domain followed by an intrinsically disordered ParE-neutralizing domain. In absence of the C-terminal IDP domain, VcParD2 crystallizes as a doughnut-shaped hexadecamer formed by the association of eight dimers. This assembly is stabilized via hydrogen bonds and salt bridges rather than hydrophobic contacts. In solution, oligomerization of the full-length protein is restricted to a stable, open 10-mer or 12-mer, likely as a consequence of entropic pressure from the IDP tails. The relative positioning of successive VcParD2 dimers mimics the arrangement of Streptococcus agalactiae CopG dimers on their operator and allows for an extended operator to wrap around the VcParD2 oligomer.

biophysics↗

bioRxiv2020

Arabidopsis Casein Kinase 2 triggers Stem Cell Exhaustion under Al Toxicity and Phosphate Deficiency Through activation of the DNA Damage Response pathway

Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses cause a strong inhibition of root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED. Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation. ONE-SENTENCE SUMMARYCasein kinase 2 and DNA damage response regulators play a pivotal role in the control of Arabidopsis root growth in response to Al toxicity and phosphate limitation.

plant biology↗

bioRxiv2020

Structure of the complete dimeric human GDAP1 core domain provides insights into ligand binding and clustering of disease mutations

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive towards classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+ homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point towards allosteric mechanisms in regulating GDAP1 oligomeric state and function.

biochemistry↗

bioRxiv2020

Structure and substrate specificity determinants of the taurine biosynthetic enzyme cysteine sulphinic acid decarboxylase

Pyridoxal 5'-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine,{gamma} -aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by non-enzymatic oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that similarly to its closest homologue, GADL1, CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.

biochemistry↗

bioRxiv2020

The TPLATE subunit is essential for structural assembly of the endocytic TSET complex

All eukaryotic cells rely on endocytosis to regulate the plasma membrane proteome and lipidome. Most eukaryotic groups, with the exception of fungi and animals, have retained the evolutionary ancient TSET complex as a regulator of endocytosis. Despite the presence of similar building blocks in TSET, compared to other coatomer complexes, structural insight into this adaptor complex is lacking. Here, we elucidate the molecular architecture of the octameric plant TSET complex (TPLATE complex/TPC) using an integrative structural approach. This allowed us to describe a plant-specific connection between the TML subunit and the AtEH/Pan1 proteins and show a direct interaction between the complex and the plasma membrane without the need for any additional protein factors. Furthermore, we identify the appendage of TPLATE as crucial for complex assembly. Structural elucidation of this ancient adaptor complex vastly advances our functional as well as evolutionary insight into the process of endocytosis. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2019

Molecular determinants of Arc oligomerization and formation of virus-like capsids

Expression of activity-regulated cytoskeleton-associated protein (Arc) is critical for long-term synaptic plasticity, memory formation, and cognitive flexibility. The ability of Arc to self-associate and form virus-like capsid structures implies functionally distinct oligomeric states. However, the molecular mechanism of Arc oligomerization is unknown. Here, we identified a 28-amino-acid region necessary and sufficient for Arc oligomerization. This oligomerization region is located within the second coil of a predicted anti-parallel coiled-coil in the N-terminal domain (NTD). Using alanine scanning mutagenesis, we found a 7-amino-acid motif critical for oligomerization and Arc-mediated transferrin endocytosis in HEK cells. Intermolecular fluorescence lifetime imaging in hippocampal neurons confirmed self-association mediated by the motif. To quantify oligomeric size, we performed a single-molecule photobleaching analysis of purified Arc wild-type and mutant. This analysis revealed a critical role for the NTD motif in the formation of higher-order Arc oligomers (30-170 molecules). Moreover, assembly of higher-order wild-type Arc oligomers was significantly enhanced by addition of GFP RNA. Purified wild-type Arc formed virus-like capsids, as visualized by negative-stain EM, and was estimated by light scattering analysis to contain 40-55 Arc units. In contrast, mutant Arc formed a homogenous dimer population as demonstrated by single-molecule TIRF imaging, size-exclusion chromatography with multi-angle light scattering analysis, small-angle X-ray scattering analysis, and single-particle 3D EM reconstruction. Thus, the dimer appears to be the basic building block for assembly. Herein, we show that the NTD motif is essential for higher-order Arc oligomerization, assembly of virus-like capsid particles, and facilitation of oligomerization by exogenous RNA. SIGNIFICANCEArc protein is rapidly expressed in neurons in response to synaptic activity and plays critical roles in synaptic plasticity, postnatal cortical developmental, and memory. Arc has diverse molecular functions, which may be related to distinct oligomeric states of the protein. Arc has homology to retroviral Gag protein and self-assembles into retrovirus-like capsid structures that are capable of intercellular transfer of RNA. Here, we identified a motif in the N-terminal coiled-coil domain of mammalian Arc that mediates higher-order oligomerization and formation of virus-like capsids. The basic building block is the Arc dimer and exogenous RNA facilitates further assembly. The identified molecular determinants of Arc oligomerization will help to elucidate the functional modalities of Arc in the mammalian brain.

biochemistry↗