Biology subjects

Uliana, F.

Publications and source records attributed to Uliana, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic identification of pH-sensing amyloid core motifs reveals a widespread mechanism for reversible protein assembly upon stress

Unlike irreversible pathological amyloids, reversible fibrils can be regulated via pH-sensing core motifs, characterized by amyloid properties unleashed upon stress-induced protonation of critical residues. Here, we combined bioinformatic predictions and an in vitro validation pipeline to search for pH-responsive, reversible amyloid core peptides in yeast and human proteomes. This approach uncovered biophysical properties distinguishing pH-sensing and constitutive amyloid cores and established reliable criteria to identify novel reversible assemblies based on sequence data. Selected full-length candidate proteins with evolutionarily conserved pH-sensing motifs were analyzed in Saccharomyces cerevisiae using fluorescence microscopy and SDS-resistance assays, revealing multiple proteins forming reversible assemblies in stationary phase. We found that protonation of a specific histidine in the amyloid core motif of the asparagine synthase Asn1 is necessary and sufficient for assembling catalytically inactive, reversible structures called cytoophidia. Interestingly, mutant cells that fail to assemble Asn1-cytoophidia show defects to recover from stationary phase, demonstrating functional relevance of pH-sensing amyloid core motifs in vivo. Taken together, we uncovered a widespread and conserved pH-sensing mechanism that regulates the reversible assembly and function of structurally diverse fibrils upon stress.

biochemistry↗

bioRxiv2026

The chaperone Jjj2 regulates nucleoporin condensation in budding yeast

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport through a selective permeability barrier established by nucleoporins (Nups) that contain intrinsically disordered phenylalanine-glycine (FG) repeats. Due to these domains, FG-Nups are prone to condensation, with a potential to transition into insoluble aggregates. How cells keep FG-Nups in a soluble, functional state during NPC assembly and within the native NPC remains poorly understood. Here, we identify the uncharacterized yeast J-domain protein Jjj2 as a Nup chaperone. Disrupting Jjj2 function or its interaction with Hsp70 chaperones triggers the accumulation of newly synthesized Nups in cytoplasmic condensates. Conversely, Jjj2 overexpression suppresses Nup condensation but also disrupts the NPC permeability barrier and is highly toxic. Overall, our data show that Jjj2, in concert with Hsp70, controls Nup phase state. This activity must be tightly regulated; while Jjj2 prevents condensation of newly produced Nups, its overactivity compromises nucleocytoplasmic compartmentalization.

cell biology↗

bioRxiv2026

Low-affinity binding motif in microtubule plus-end condensates specializes microtubule function

The microtubule plus-end tracking proteins (+TIPs) CLIP-170/Bik1 and EB/Bim1 form a condensate, the +TIP body, at the plus-end of most microtubules in vivo. Remarkably, however, these +TIP bodies typically impart different dynamics and interaction profiles to distinct microtubules, according to their cellular function. The molecular mechanisms underlying the functional versatility of the +TIP body are unknown. Here, we show that the +TIP Kar9 utilizes repeats of a lysine-aspartate-lysine (KDK)-centered short linear motif (SLiM) to interact with Bik1 on a restricted subset of cytoplasmic microtubules during yeast mitosis. Furthermore, these multivalent Kar9-Bik1 interactions tune the material behavior of the +TIP body to specify proper microtubule function. Indicating that KDK serves as generic Bik1-interaction motif, similar motifs are also present in Kip2, where they mediate Bik1-dependent recruitment of Kip2 to the +TIP body. Together, our study provides insights into how low-affinity Bik1 interactors diversify microtubule function by locally specializing the content and behavior of +TIP bodies.

Cell Biology↗

bioRxiv2026

Cell wall remodeling is required for crowding homeostasis during cell growth in S. cerevisiae

The cytoplasm is a densely crowded environment that plays essential roles in intracellular functions. Its biophysical properties can be markedly altered in response to environmental cues and changes in cellular state. Recent studies have shown that excessive growth leads to cytoplasmic dilution, contributing to cellular dysfunction. However, the molecular mechanisms regulating biophysical properties during excessive growth remain poorly understood. Here, we demonstrate that cell wall remodeling is crucial to maintain cytoplasmic crowding homeostasis during cell growth. Live-cell imaging and phosphoproteomic analysis showed that Mpk1, a component of Cell Wall Integrity (CWI) pathway, accumulates in the nucleus and its activity progressively increases as cells enlarge. Mpk1 is required to restrict cell volume expansion and maintain cytoplasmic crowding by adjusting cell wall thickness and mechanical resistance. Moreover, Mpk1 is essential for the survival of enlarged cells upon cell cycle re-entry. In addition to Mpk1, we identified multiple key components of the CWI pathway that are required to maintain physiological state of enlarged cells. Together, these findings establish the CWI signaling pathway as a key regulator of cell size homeostasis and cytoplasmic biophysical properties, providing a mechanistic link between cell wall remodeling and intracellular properties.

Cell Biology↗

bioRxiv2026

Phase separation behavior of TDP-43 governs its protein interactome and regulation of altern

TDP-43 is a nuclear RNA-binding protein that regulates RNA metabolism, including alternative splicing. Its aggregation is a major pathological hallmark of several neurodegenerative diseases. TDP-43 undergoes phase separation (PS) and this condensation behavior may be linked to aggregate formation. Whether and how PS governs TDP-43 RNA regulatory functions remains poorly understood. Here we utilized rationally designed mutations in the TDP-43 low complexity domain to tune TDP-43 PS, yielding a panel of TDP-43 variants with reduced propensity to form condensates (PS-deficient), and a panel forming irreversible, undynamic condensates (solid-like) in vitro and in cells. Two complementary interactomics approaches identified PS-dependent interactions between TDP-43 and key RNA regulatory factors, including splicing regulators and the RNA helicase UPF1, which show increased interactions with solid-like variants. Our results highlight that TDP-43 PS regulates RNA and protein homeostasis by modulating a subset of TDP-43-dependent alternative splicing events and by reshaping interactions with RNA regulatory factors.

biochemistry↗

bioRxiv2026

Molecular determinants underlying substrate receptor specificity of human CRL4B E3 ubiquitin ligase

The vertebrate CRL4 family of Cullin-RING E3 ubiquitin ligases is distinguished from other cullin-based ligases by the presence of two highly homologous paralogs, CUL4A and CUL4B. Both CRL4 complexes use the DDB1 subunit to recruit dedicated and interchangeable substrate receptors called DCAFs, but the underlying mechanisms guiding DCAF specificity for CUL4B or CUL4A remain poorly understood. Here, we performed structural and biochemical analyses of the CRL4BLIS1 complex and identified two molecular determinants for CUL4B-specific DCAFs. First, we discovered that the unique CUL4B N-terminal extension directly binds CUL4B-specific DCAFs, enhancing their complex formation. This direct interaction can be modulated by phosphorylation, adding the possibility for spatiotemporal regulation. Second, the cryo-EM model of the CRL4BLIS1 complex identified a novel interface on the DDB1 subunit which promotes LIS1 recruitment. Quantitative affinity measurements and mutational analysis confirmed that this DDB1 interface is generally important for recruiting CUL4B-specific DCAFs including WDR1 and BRWD1, but not for CUL4A-specifc DCAFs, such as DCAF8. Together, our study identifies molecular determinants and unexpected interfaces on CRL4 components that dictate preference for DCAF recruitment. Graphical Abstract showing how the two CRL4 complexe s, namely CRL4A and CRL4B, recruit their DCAFs O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/711546v1_ufig1.gif" ALT="Figure 1000"> View larger version (39K): org.highwire.dtl.DTLVardef@10e476eorg.highwire.dtl.DTLVardef@73a405org.highwire.dtl.DTLVardef@1571a1org.highwire.dtl.DTLVardef@1d70946_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

Growth-induced compressive forces regulate diverse biological processes ranging from development to tumor progression, but the underlying molecular mechanisms remain poorly understood. Here, we found that compressive mechanical cues activate the MAP kinase extracellular signal-regulated kinase 5 (ERK5) at the cell periphery, inducing invasive traits in epithelial cells. Using a phospho-proteomic approach combined with an RNAi-based validation screen, we identified several previously unrecognized ERK5 effectors important for cell-cell adhesion and actin organization, among them the myosin light chain (MLC) phosphatase subunit Myosin Phosphatase-Targeting Subunit 1 (MYPT1). Our results demonstrate that ERK5 regulates MYPT1, which in turn inhibits myosin at the cell periphery. ERK5 signaling counteracts Rho-associated protein kinase (ROCK), thereby reducing peripheral actomyosin tension. This relaxation destabilizes cell-cell contacts and promotes cell migration in response to mechanical compression. Together, these findings identify an ERK5-mediated response mechanism activated by compressive force that triggers cell invasion, highlighting ERK5 therapeutic potential.

cell biology↗

bioRxiv2025

Dystonia-associated Torsins sustain CLCC1 function to promote membrane fusion of the nuclear envelope for NPC biogenesis

DYT1 early-onset dystonia is a severe, incurable disorder of the central nervous system caused by mutations in the gene encoding Torsin1A (Tor1A, DYT1). Torsins are ER-resident AAA+-ATPases implicated in lipid metabolism, nuclear pore complex (NPC) biogenesis, and lipoprotein secretion, yet their molecular function that underlies the disease pathology has remained incompletely understood. Here, we have utilized Drosophila melanogaster and human somatic cells as experimental models to shed light on their mode-of-action. Fly germ cells lacking dTorsin are arrested in development and display defects in the final steps of NPC biogenesis due to a failure in fusion of the inner and outer nuclear membranes. Using proximity labelling of Torsin1A in human cells, we identify the conserved membrane protein chloride channel CLIC-like protein 1 (CLCC1) as a novel Torsin binding partner. Absence of human CLCC1 or its Drosophila homolog dClcc1 phenocopied the membrane fusion defects at NPC assembly sites observed upon Torsin deletion. Furthermore, CLCC1 is enriched at arrested fusion sites, suggesting it to be a candidate for the elusive NE membrane fusogen. Importantly, CLCC1/dClcc1 overexpression is sufficient to rescue NPC biogenesis and developmental defects associated with Torsin-loss-of-function. Taken together, our data suggest that Torsin-regulated CLCC1 activity drives membrane fusion during NPC biogenesis and reveal that modulating CLCC1 expression is a promising therapeutic prospect for DYT1 dystonia.

cell biology↗

bioRxiv2025

Disruption of nucleolar integrity triggers cellular quiescence through organelle rewiring and secretion

The nucleolus is the largest membraneless nuclear organelle and a critical regulator of growth and stress responses, comprised of over 600 proteins involved in ribosome biogenesis. However, how the nucleolar function and architecture coordinate cell-wide adaptive programs remains unclear. Here, we combined multi-omics profiling with functional genomics to define the cellular consequences of nucleolar disruption. We identify a stress response pathway connecting the nucleolus to the Golgi apparatus, endo-lysosomal trafficking, and cellular secretion (NuGETS). Chronic nucleolar defects activate a TP53-dependent transcriptional program that promotes organelle expansion, enhances secretory activity, and induces cell cycle exit with prolonged quiescence and partial epithelial-to-mesenchymal transition (pEMT) phenotypes. A genome-wide CRISPRi screen uncovered 400 regulators of secretory and quiescent states upon nucleolar stress. These pathways are enriched in cancer progression associated with ribosomopathies. Our findings therefore link nucleolar stress responses and cancer development, revealing cell-wide regulatory mechanisms that safeguard survival when nucleolar function is compromised.

cell biology↗

bioRxiv2025

Direct binding of TDP-43 and Tau drives their co-condensation, but suppresses Tau fibril formation and seeding

Neuronal Tau aggregates are a hallmark of Alzheimers disease (AD), but more than half of the patients exhibit additional TDP-43 inclusions and some have co-aggregates of both proteins. The presence of Tau/TDP-43 co-pathology is associated with increased disease severity, although the causal relationship remains unclear. Here we demonstrate that Tau and TDP-43 mutually promote each others condensation through direct interaction in vitro, forming irregularly shaped or multiphasic co-condensates with lower TDP-43 mobility, but higher Tau dynamics. While Tau promotes TDP-43 aggregation in vitro, TDP-43 suppresses formation of Tau fibrils and instead causes formation of oligomeric Tau and Tau/TDP-43 species. These co-assemblies hinder Tau seeding in a biosensor assay specific for proteopathic Tau seeds. Consistent with this data, SarkoSpin extracts from AD brains with Tau/TDP-43 co-pathology exhibit reduced Tau seeding compared to Tau-only AD brains. In contrast, patient-derived extracts from AD brains with Tau/TDP-43 co-pathology are highly potent in seeding TDP-43 neoaggregates in a TDP-43 reporter cell line. Our results suggest that direct interaction of TDP-43 and Tau may suppress Tau pathology, while promoting TDP-43 pathology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/662960v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ed5dfaorg.highwire.dtl.DTLVardef@b4ef07org.highwire.dtl.DTLVardef@b8dbe8org.highwire.dtl.DTLVardef@6d9b49_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

PKA regulates stress granule maturation to allow timely recovery after prolonged starvation

Cells have evolved multiple strategies to survive environmental stress conditions. This includes the formation of membrane-less cytoplasmic ribonucleoprotein structures called stress granules (SGs) that sequester and protect mRNAs encoding many housekeeping genes. SGs are not static biomolecular condensates but transform into solid states during a maturation phase. Although SG maturation is a hallmark of many neurodegenerative pathologies, little is known about the mechanisms and physiological relevance underlying the maturation process. Here we show that yeast SGs mature into a solid-like state during long-term stationary phase stress, which delays SG disassembly and cell cycle restart. Profiling of phosphorylation sites during stationary phase revealed that SG maturation is driven by protein kinase A (PKA)-dependent phosphorylation of the SG proteome. Indeed, upon stationary phase the catalytic PKA subunits condense in SGs where they maintain kinase activity, while during this time cytoplasmic PKA is inhibited. PKA-mediated phosphorylation of key SG components, like the pyruvate kinase Cdc19, is necessary and sufficient for its timely accumulation in SGs, where Cdc19 assembles into amyloid-like structures. Importantly, inhibiting PKA during long-term stationary phase prevents SG maturation, delaying ordered re-start of cell growth after re-feeding. Taken together, these results describe a SG maturation mechanism, selectively activated during chronic stress, that preserves SG integrity and promotes cell survival. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/663161v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@19c4d82org.highwire.dtl.DTLVardef@a4cf27org.highwire.dtl.DTLVardef@1c2577org.highwire.dtl.DTLVardef@1dfe84f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIStress granules mature during chronic stress such as long-term stationary phase C_LIO_LIPhosphorylation of SG components by PKA is a hallmark of SG maturation C_LIO_LIPhosphorylation of Cdc19 by PKA promotes its maturation into an amyloid-like state C_LIO_LISG maturation acts as a timer for recovery after chronic stress C_LI

cell biology↗

bioRxiv2024

The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII

Although the connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain unclear. Available data support that the burst of cytokines and bradykinin, observed in some COVID-19 patients, sustains systemic inflammation and the hypercoagulant state, thus increasing thrombotic risk. Here we show that the SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma from healthy donors increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factors zymogens VII and XII at their physiological activation sites, i.e. Arg152-Ile153 bond for FVII and Arg353-Val354 bond for FXII, where FVII and FXII are strategically positioned at the very beginning of the extrinsic or intrinsic pathways of blood coagulation. These findings are not compatible with the substrate specificity of the protease known so far, displaying a prevalence for a Gln-residue in P1 and a hydrophobic amino acid in P2 position. This apparent discrepancy was resolved by High Throughput Protease Screen assay, unveiling an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by Hydrogen-Deuterium Exchange Mass spectrometry analysis of Arg-containing inhibitors binding to Mpro and by enzymatic assays showing that the protease can cleave peptide substrates containing Arg in P1. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.

biochemistry↗

bioRxiv2024

Stm1 regulates Ifh1 activity revealing crosstalk between ribosome biogenesis and ribosome dormancy

Ribosome abundance in changing environments is governed by biogenesis and degradation, but the underlying mechanisms regulating these opposing processes remain unknown. Here we show that Suppressor of Tom1 (Stm1), a dormancy factor protecting cytosolic ribosomes during starvation, has an unexpected function to promote ribosome biogenesis during exponential growth conditions. Indeed, Stm1 transiently localizes to the nucleolus and engages with pre-ribosomal particles. Stm1 upregulates transcription of ribosomal protein genes by directly binding the activation domain (AD) of the transcription factor Ifh1. These novel Stm1 functions confer rapamycin-sensitivity and are mediated by its C-terminal intrinsically disordered region (IDR), which is dispensable for ribosome hibernation. We conclude that Stm1 regulates ribosome homeostasis linking ribosome biogenesis and ribosome dormancy.

cell biology↗

bioRxiv2024

Phase separation of a microtubule plus-end tracking protein into a fluid fractal network

Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based cellular processes, and it has been recently proposed that they function as liquid condensates. However, the formation process and internal organization of +TIP condensates are poorly understood. Here, we have investigated the phase separation of the CLIP-170 family member Bik1, a key +TIP implicated in budding yeast cell division. We found that Bik1 is a rod-shaped dimer whose conformation is dominated by its central coiled-coil domain. Liquid condensation is accompanied by Bik1 conformational rearrangements, leading to a 2-3-fold rise in interactions between the proteins folded and disordered domains. In contrast to classical liquids, the supramolecular structure of the Bik1 condensate is heterogeneous, with a fractal structure of protein-rich and protein-free domains. This observation provides structural evidence in support of recent models of biomolecular condensates based on percolation. More broadly, our results provide insights into the structure, dynamic rearrangement, and organization of a complex, multidomain protein in its dilute and condensed phases. Our experimental framework can be extended to other biomolecular condensates, including more intricate +TIP networks.

biochemistry↗

bioRxiv2023

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate

The association of genomic loci to the nuclear periphery is proposed to facilitate cell-type specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting [~]1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein, Stonewall (Stwl), as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwls known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

cell biology↗

bioRxiv2023

The hGID GID4 E3 ubiquitin ligase complex targets ARHGAP11A to regulate cell migration

The human CTLH/GID (hGID) complex emerged as an important E3 ligase regulating multiple cellular processes, including cell cycle progression and metabolic activity. However, the range of biological functions controlled by hGID remains unexplored. Here, we show that the hGID substrate receptor GID4 regulates cell growth and migration. Biochemical and cellular assays combined with proximity-dependent biotinylation (BioID2) revealed that the hGIDGID4 E3-ligase targets the Rho-GAP ARHGAP11A for degradation. Depletion of GID4 or impeding the GID4 substrate binding pocket impairs motility and directed cell movement, whereas knockdown of ARHGAP11A significantly restores the cell migration defect. We found that GID4 controls cell migration by degrading ARHGAP11A thereby preventing its accumulation at the cell periphery where it inactivates RhoA activity. Together, we identified a unique function for GID4, as well as a wide range of substrate profiles beyond Pro/N-degron motifs, which pave the way for deciphering additional pathways regulated by hGID E3 ligase activity through its GID4 substrate receptor.

biochemistry↗

bioRxiv2023

Multi-tissue proteomics identifies a link between satellite DNA organization and transgenerational transposon repression in Drosophila

Non-coding satellite DNA repeats are abundant at the pericentromeric heterochromatin of eukaryotic chromosomes. During interphase, sequence-specific DNA-binding proteins cluster these repeats from multiple chromosomes into nuclear foci known as chromocenters. Despite the pivotal role of chromocenters in cellular processes like genome encapsulation and gene repression, the associated proteins remain incompletely characterized. Here, we use two satellite DNA-binding proteins, D1 and Prod, as baits to characterize the chromocenter-associated proteome in Drosophila embryos, ovaries, and testes through quantitative mass spectrometry. We identify D1- and Prod-associated proteins, including known heterochromatin proteins as well as proteins previously unlinked to satellite DNA or chromocenters, thereby laying the foundation for a comprehensive understanding of cellular functions enabled by satellite DNA repeats and their associated proteins. Interestingly, we find that multiple components of the transposon-silencing piRNA pathway are associated with D1 and Prod in embryos. Using genetics, transcriptomics, and small RNA profiling, we show that flies lacking D1 during embryogenesis exhibit transposon expression and gonadal atrophy as adults. We further demonstrate that this gonadal atrophy can be rescued by mutating the checkpoint kinase, Chk2, which mediates germ cell arrest in response to transposon mobilization. Thus, we reveal that a satellite DNA-binding protein functions during embryogenesis to silence transposons, in a manner that is heritable across later stages of development.

cell biology↗

bioRxiv2023

DIP-MS: A novel ultra-deep interaction proteomics for the deconvolution of protein complexes

Most, if not all, proteins are organized in macromolecular assemblies, which represent key functional units regulating and catalyzing the majority of cellular processes in health and disease. Ever-advancing analytical capabilities promise to pinpoint lesions in proteome modularity driving disease phenotypes. Affinity purification of the protein of interest combined with LC-MS/MS (AP-MS) represents the method of choice to identify interacting proteins. The composition of complex isoforms concurrently present in the AP sample can however not be resolved from a single AP-MS experiment but requires computational inference from multiple time-and resource-intensive reciprocal AP-MS experiments. In this study we introduce Deep Interactome Profiling by Mass Spectrometry (DIP-MS) which combines affinity enrichment with BN-PAGE separation, DIA mass spectrometry and deep-learning-based signal processing to resolve complex isoforms sharing the same bait protein in a single experiment. We applied DIP-MS to probe the organisation of the human prefoldin (PFD) family of complexes, resolving distinct PFD holo- and sub-complex variants, complex-complex interactions and complex isoforms with new subunits that were experimentally validated. Our results demonstrate that DIP-MS can reveal proteome modularity at unprecedented depth and resolution and thus represents a critical steppingstone to relate a proteome state to phenotype in both healthy and diseased conditions.

systems biology↗