Biology subjects

Uliana, F.

Publications and source records attributed to Uliana, F..

27 records · Page 2Linked to original sources

bioRxiv2022

Cell cycle progression defects and impaired DNA damage signaling drive enlarged cells into senescence

Cellular senescence plays an important role in development, ageing, and cancer biology. Senescence is associated with increased cell size, but how this contributes to permanent cell cycle exit is poorly understood. Using reversible G1 cell cycle arrests combined with growth rate modulation, we examined the effects of excess cell size on cell cycle progression in human cells. We show that enlarged cells paradoxically have high levels of G1/S regulators relative to cells that were maintained at physiological size but also induce p21, which restrains cell cycle entry and protects against cell division failure. Furthermore, we find that enlarged cells bear an increased propensity for DNA breakage and concomitant DNA damage repair defects that are established during G1. Based on these observations, we propose that impaired DNA damage repair pathways prime enlarged cells for persistent replication-acquired damage, ultimately leading to catastrophic cell cycle failure and permanent cell cycle exit.

cell biology↗

bioRxiv2022

Systematic dissection of phosphorylation-dependent YAP1 complex formation elucidates a key role for PTPN14 in Hippo signal integration

Cellular signaling relies on the temporal and spatial control of the formation of transient protein complexes by post-translational modifications, most notably by phosphorylation. While several computational methods have been developed to predict the functional relevance of phosphorylation sites, assessing experimentally the interdependency between protein phosphorylation and protein-protein interactions (PPIs) remains a major challenge. Here, we describe an experimental strategy to establish interdependencies between specific phosphorylation events and complex formation. This strategy is based on three main steps: (i) systematically charting the phosphorylation landscape of a target protein; (ii) assigning distinct proteoforms of the target protein to different protein complexes by electrophoretic separation of native complexes (BNPAGE) and protein/phopho correlation profiling; and (iii) genetically deleting known regulators of the target protein to identify which ones are required for given proteoforms and complexes. We applied this strategy to study phosphorylation- dependent modulation of complexes containing the transcriptional co-regulator YAP1. YAP1 is highly phosphorylated and among the most extensively connected proteins in the human interactome. It functions as the main signal integrator and effector protein of the Hippo pathway which controls organ size and tissue homeostasis. Using our workflow, we could identify several distinct YAP1 proteoforms specifically associated with physically distinct complexes and infer how their formation is affected by known Hippo pathway members. Importantly, our findings suggest that the tyrosine phosphatase PTPN14 controls the co-transcriptional activity of YAP1 by regulating its interaction with the LATS1/2 kinases. In summary, we present a powerful strategy to establish interdependencies between specific phosphorylation events and complex formation, thus contributing to the "functionalization" of phosphorylation events and by this means provide new insights into Hippo signaling.

systems biology↗

bioRxiv2022

An amphipathic helix in Brl1 is required for membrane fusion during nuclear pore complex biogenesis in S. cerevisiae

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-ET analyses reveal multi-layered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.

cell biology↗

bioRxiv2022

Condensation of a nuclear mRNA export factor regulates mRNA transport during stress

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that upon glucose withdrawal the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and to control gene expression during stress. HighlightsO_LIThe nuclear poly(A)-binding protein Nab2 forms RNA-containing condensate-like structures upon glucose starvation and upon acute cellular depletion of the DEAD-box ATPase Dbp5 C_LIO_LIThe Nab2 multimerization interface but not the intrinsically disordered regions (IDRs) are essential for condensation in vitro and in vivo C_LIO_LIGlucose stress leads to poly(A) RNA retention in the nucleus, which is affected by the state of the Nab2 condensate C_LI

cell biology↗

bioRxiv2021

Rapid profiling of protein complex re-organization in perturbed systems

Protein complexes constitute the primary functional modules of cellular activity. To respond to perturbations, complexes undergo changes in their abundance, subunit composition or state of modification. Understanding the function of biological systems requires global strategies to capture this contextual state information on protein complexes and interaction networks. Methods based on co-fractionation paired with mass spectrometry have demonstrated the capability for deep biological insight but the scope of studies using this approach has been limited by the large measurement time per biological sample and challenges with data analysis. As such, there has been little uptake of this strategy beyond a few expert labs into the broader life science community despite rich biological information content. We present a rapid integrated experimental and computational workflow to assess the re-organization of protein complexes across multiple cellular states. It enables complex experimental designs requiring increased sample/condition numbers. The workflow combines short gradient chromatography and DIA/SWATH mass spectrometry with a data analysis toolset to quantify changes in complex organization. We applied the workflow to study the global protein complex rearrangements of THP-1 cells undergoing monocyte to macrophage differentiation and a subsequent stimulation of macrophage cells with lipopolysaccharide. We observed massive proteome organization in functions related to signaling, cell adhesion, and extracellular matrix during differentiation, and less pronounced changes in processes related to innate immune response induced by the macrophage stimulation. We therefore establish our integrated differential pipeline for rapid and state-specific profiling of protein complex organization with broad utility in complex experimental designs.

systems biology↗

bioRxiv2021

Novel biochemical, structural and systems insights into inflammatory signaling revealed by contextual interaction proteomics

Protein-protein interactions (PPI) represent the main mode of the proteome organization in the cell. In the last decade, several large-scale representations of PPI networks have captured generic aspects of the functional organization of network components, but mostly lack the context of cellular states. However, the generation of contextual representations of PPI networks is essential for structural and systems-level modeling of biological processes and remains an unsolved challenge. In this study we describe an integrated experimental/computational strategy to achieve a contextualized modeling of PPI. This strategy defines the composition, stoichiometry, spatio-temporal organization and cellular requirements for the formation of target assemblies. We used this approach to generate an integrated model of the formation principles and architecture of a large signalosome, the TNF-receptor signaling complex (TNF-RSC). Overall, we show that the integration of systems- and structure-level information provides a generic, largely unexplored link between the modular proteome and cellular function. Significance StatementIn this work, we propose a critical shift in the way we analyze, and think the study of, protein-protein interactions (PPI), and present an experimental and computational framework to model them in the cellular context. We applied this framework to the signalosome tumor necrosis factor receptor signaling complex (TNF-RSC), and generated an integrated model of its formation and architecture that provides new insights and resolved controversies regarding its organization and regulation. To achieve a contextual modelling of PPIs, we first optimized and developed, and then combined, approaches to map the composition of a target complex, its absolute stoichiometry, its spatial organization and assembly/disassembly dynamics, its temporal dependence on signaling, and its reliance on cellular resources.

systems biology↗

bioRxiv2021

Human -Synuclein Inhibits Platelets Aggregation in vitro by Interfering with the -Thrombin/Protease-Activated Receptor 1 Functional Axis

-Synuclein (Syn) is a small (140 amino acids) disordered, acidic (pI: 4.7) protein, highly conserved in vertebrates and implicated in the pathogenesis of Parkinsons disease (PD), a neurodegenerative disease characterized by the deposition of Syn amyloid fibrils in dopaminergic neurons. Beyond the central nervous system, significant expression of Syn has also been measured in the blood (~1 M), where platelets are the main cellular hosts of Syn. Although the pathological implication of Syn in PD is widely accepted, the physiological role of blood Syn is still elusive. Starting from the notion that platelets are either the major cellular reservoir of Syn in the blood and, concomitantly, act as key players in hemostasis, being activated also by -thrombin (T) via cleavage of protease-activated receptors (PARs), we decided to investigate the possibility that Syn could modulate platelet activation by interfering with the T-PAR functional axis. Using multiple electrode aggregometry, i.e. a fast and specific platelet-function-testing method, as well as steady-state fluorescence spectroscopy, surface plasmon resonance, and fluorescence microscopy, we show here that monomeric Syn functions as a negative regulator of T-mediated platelets activation. Syn acts either directly, via competitive inhibition of PAR1 activation by T and TRAP6 agonist, and indirectly, by scavenging T on the platelet plasma membrane. A simple electrostatic model of Syn platelet antiaggregating effect is proposed and the possible role of the protein at the interplay of amyloidosis and thrombosis is discussed.

biochemistry↗

bioRxiv2020

Mapping specificity, entropy, allosteric changes and substrates in blood proteases by a high-throughput protease screen

Proteases are among the largest protein families in eukaryotic phylae with more than 500 genetically encoded proteases in humans. By cleaving a wide range of target proteins, proteases are critical regulators of a vast number of biochemical processes including apoptosis and blood coagulation. Over the last 20 years, knowledge of proteases has been drastically expanded by the development of proteomic approaches to identify and quantify proteases and their substrates. In spite of their merits, some of these methods are laborious, not scalable or incompatible with native environments. Consequentially, a large number of proteases remain poorly characterized. Here, we introduce a simple proteomic method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter and their analysis in a mass spectrometer. The method is significantly faster, cheaper, technically less demanding, easily multiplexed and produces accurate protease fingerprints in near-native conditions. By using the blood cascade proteases as a case study we obtained protease substrate profiles of unprecedented depth that can be reliably used to map specificity, entropy and allosteric changes of the protease and to design fluorescent probes and predict physiological substrates. The native protease characterization method is comparable in performance, but largely exceeds the throughput of current alternatives.

biochemistry↗

bioRxiv2020

Systematic protein complex profiling and differential analysis from co-fractionation mass spectrometry data

Protein complexes, macro-molecular assemblies of two or more proteins, play vital roles in numerous cellular activities and collectively determine the cellular state. Despite the availability of a range of methods for analysing protein complexes, systematic analysis of complexes under multiple conditions has remained challenging. Approaches based on biochemical fractionation of intact, native complexes and correlation of protein profiles have shown promise, for instance in the combination of size exclusion chromatography (SEC) with accurate protein quantification by SWATH/DIA-MS. However, most approaches for interpreting co-fractionation datasets to yield complex composition, abundance and rearrangements between samples depend heavily on prior evidence. We introduce PCprophet, a computational framework to identify novel protein complexes from SEC-SWATH-MS data and to characterize their changes across different experimental conditions. We demonstrate accurate prediction of protein complexes (AUC >0.99 and accuracy around 97%) via five-fold cross-validation on SEC-SWATH-MS data, show improved performance over state-of-the-art approaches on multiple annotated co-fractionation datasets, and describe a Bayesian approach to analyse altered protein-protein interactions across conditions. PCprophet is a generic computational tool consisting of modules for data pre-processing, hypothesis generation, machine-learning prediction, post-prediction processing, and differential analysis. It can be applied to any co-fractionation MS dataset, independent of separation or quantitative LC-MS workflow employed, and to support the detection and quantitative tracking of novel protein complexes and their physiological dynamics.

systems biology↗