Biology subjects

Cianferani, S.

Publications and source records attributed to Cianferani, S..

13 recordsLinked to original sources

bioRxiv2026

The R2T(P) complex orchestrates the SHQ1 driven early steps of box H/ACA snoRNP maturation

The chaperones RuvBL1 and RuvBL2 are members of the AAA+ ATPase family and participate in diverse cellular processes, including DNA repair, transcriptional regulation, and assembly of macromolecular complexes such as snoRNPs. The biogenesis of box H/ACA snoRNPs additionally requires the assembly factor SHQ1. These protein-RNA complexes are essential for ribosome biogenesis and telomerase stability and are linked to diseases such as dyskeratosis congenita and cancer. Despite detailed knowledge of mature complexes, their assembly mechanisms and how they can be modulated remain unclear. We characterize a trimeric interaction between SHQ1 and RuvBL1:RuvBL2, providing insight into early maturation of the protein-only precursor of box H/ACA snoRNPs. SHQ1 binds the flexible domain II of RuvBL1:RuvBL2, corresponding to the dodecamerization interface, suggesting disruption of this interface and promotion of hexamer formation. We further purified a complex containing RuvBL1:RuvBL2, SHQ1, and DKC1, the catalytic component of H/ACA snoRNPs and a client of SHQ1. This demonstrates that SHQ1 and DKC1 can simultaneously associate with RuvBL1:RuvBL2, potentially facilitating DKC1 release and subsequent snoRNA binding. Additionally, we identified a direct interaction between SHQ1 and RPAP3, a co-chaperone of RuvBL1:RuvBL2 (within the R2TP). Hence, RPAP3 may be responsible for recruiting SHQ1:DKC1 to hexameric RuvBL1:RuvBL2 and/or assist in the AAA+ mediated dissociation of the dimer. Since SHQ1 shares a domain with PIH1D1, an integral member of the R2TP complex, our findings suggest that early H/ACA snoRNP maturation may involve the R2T instead of the previously proposed R2TP complex.

biophysics↗

bioRxiv2025

FAIMS-GPF XL-MS: crosslinking-mass spectrometry based on gas-phase fractionation

Protein-protein interactions (PPIs) underpin nearly all cellular processes; therefore, mapping PPI and protein-protein association networks is critical for understanding how biological systems function in health and disease. However, many functionally relevant PPIs are transient and mediated by weak affinity interactions, making them challenging to detect. Using chemical cross-linking together with mass spectrometry (XL-MS) has emerged as an important category of methods for mapping PPIs, including those that are more dynamic and transient, although XL-MS workflows face limitations which hinder their routine use, especially in proteomic platforms. Here, to address these limitations, we developed a XL-MS workflow based on gas-phase fractionation (GPF) using high-field asymmetric waveform ion mobility spectrometry (FAIMS). Our optimized FAIMS-GPF XL-MS protocols exhibited improved performance when handling both low-complexity samples, such as purified Cdk7-Activating Kinase (CAK) heterotrimer, and high-complexity cross-linked HeLa lysates. In HeLa lysate, we identified 1,278 cross-links accounting for 213 PPIs without any in-solution fractionation, starting from less than 20 {micro}g of sample. The method also proved effective for analyzing low-abundance, high-complexity samples such as spliceosomes. Overall, FAIMS-GPF enhanced the detection of cross-linked peptides and improved PPI coverage, thus offering a scalable, high-sensitivity solution for XL-MS integration with structural biology and functional proteomics applications.

systems biology↗

bioRxiv2025

Identification of uranyl-binding proteins in Arabidopsis thaliana cells exposed to uranium: Insights from a metalloproteomic analysis and characterization of Glycine-Rich RNA-binding Protein 7 (GRP7)

Uranium (U) is a naturally occurring radionuclide, chemotoxic for living organisms. To identify proteins that could be cellular targets for U in plants, we used metalloproteomic approaches combining column chromatographic fractionation analyses, protein identification by high-resolution mass spectrometry shotgun proteomics, and metal quantification by induced coupled plasma mass spectrometry. We identified 57 candidate proteins for uranyl (U(VI)) binding in cultured Arabidopsis thaliana cells. One of these proteins, the Glycine-Rich RNA-binding Protein 7 (GRP7) is a RNA-binding protein involved in various developmental processes and responses to biotic and abiotic stress. Recombinant GRP7 was purified from overproducing bacteria and subjected to further biochemical characterization. First, we showed that GRP7 binds U(VI) with a 1:2 (protein:metal) stoichiometry in vitro. Next, we analyzed its structural properties by solution-state nuclear magnetic resonance spectroscopy. This allowed us to gain insight into the molecular dynamics of the protein-metal interaction and to identify residues involved in U(VI)-binding in the two sites. Finally, we observed that U(VI) binding interferes with nucleic acid binding to the protein RNA-binding domain, suggesting that U(VI) binding to GRP7 contributes to U toxicity in plants. HIGHLIGHTSO_LI57 candidate uranyl-binding proteins were identified from Arabidopsis cells exposed to uranyl, of which the RNA-binding protein GRP7. C_LIO_LIIn vitro, recombinant GRP7 binds 2 uranyl ions within the RNA recognition motif domain. C_LIO_LIAmino acid residues involved in uranyl binding in both binding sites were identified. C_LIO_LICompetition between specific oligonucleotide and uranyl for binding suggests the implication of GRP7 in uranium toxicity, as a cellular target. C_LI

biochemistry↗

bioRxiv2025

Designing new natural-mimetic phosphatidic acid: aversatile and innovative synthetic strategy forglycerophospholipid research

Glycerophospholipids (GPLs) play important roles in cellular compartmentalization and signaling. Among them, phosphatidic acids (PA) exist as many distinct species depending on acyl chain composition, each one potentially displaying unique signaling function. Although the signaling functions of PA have already been demonstrated in multiple cellular processes, the specific roles of individual PA species remain obscure due to a lack of appropriate tools. Indeed, current synthetic PA analogues fail to preserve all the functions of natural PA. To circumvent these limitations, we developed a novel synthetic approach to produce PA analogues without compromising structural integrity of acyl chains. Moreover, addition of a clickable moiety allowed flexible grafting of different molecules to PA analogues for various biological applications. Hence, this innovation also provides powerful tools to investigate specific biological activities of individual PA species, with potential applications in unraveling complex GPL-mediated signaling pathways.

neuroscience↗

bioRxiv2025

R2TP-like Quaternary Chaperones: a comprehensive overview to understand the dynamic R2SP complex

The human R2SP complex belongs to the R2TP-like quaternary chaperone family and consists of RUVBL1, RUVBL2, SPAG1 and PIH1D2. R2SP is crucial for the correct assembly of motile cilia (SPAG1 null mutations cause Primary Ciliary Dyskinesia) and the organization of the synaptic zone. RUVBL1/2 ATPases are the powerhouse of this molecular machinery, while SPAG1 and PIH1D2 would be adaptors that interact with specific clients to promote their quaternary assembly. Despite these functional data, little is known about the structure of R2SP and the precise mode of action of these R2TP-like complexes. We have combined biochemical and structural approaches (NMR, structural mass spectrometry and cryo-EM) to investigate the 3D organization of the human R2SP complex, its mode of assembly and ATPase activity. Our study reveals a three-dimensional structure similar to that of the canonical R2TP complex, but also highlights differences in the mode of action of its RUVBL1/2 ATPase core as well as the binding of its adaptors SPAG1 and PIH1D2.

biophysics↗

bioRxiv2024

Deciphering molecular determinants of GPCR-G protein receptor interactions by complementary integrative structural biology methods

Many physiological processes are dependent on G protein-coupled receptors (GPCRs), the biggest family of human membrane proteins and a significant class of therapeutic targets. Once activated by external stimuli, GPCRs use G proteins and arrestins as transducers to generate second messengers and trigger downstream signaling, leading to diverse signaling profiles. The G protein-coupled bile acid receptor 1 (GPBAR1, also known as Takeda G protein-coupled receptor 5, TGR5) is a class A bile acid membrane receptor that regulates energy homeostasis and glucose and lipid metabolism. GPBAR1/G protein interactions are implicated in the prevention of diabetes and the reduction of inflammatory responses, making GPBAR1 a potential therapeutic target for metabolic disorders. Here, we present an integrated structural biology approach combining hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) to identify the molecular determinants of GPBAR1 conformational dynamics upon G protein binding. Thanks to extensive optimization of both HDX-MS and cryo-EM workflows, we achieved over 99% sequence coverage along with a 2.5-[A] resolution structure, both of which are state-of-the-art and solely obtained for complete GPCR complexes. Altogether, our results provide information on the under-investigated GPBAR1 binding mode to its cognate G protein, pinpointing the synergic and powerful combination of higher (cryo-EM) and lower (HDX-MS) resolution structural biology techniques to dissect GPCR/G protein binding characteristics. Short statementThis work highlights the utility of integrating cryo-EM and HDX-MS for studying large multiprotein complexes such as GPCR/G protein complexes. Cryo-EM offers high-resolution structural details, while HDX-MS reveals the dynamic conformational changes during assembly, providing a comprehensive structural view of difficult-to-study membrane protein systems.

biophysics↗

bioRxiv2023

Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level

Mass photometry (MP) is a versatile, fast and low sample-consuming biophysical technique that gained interest in structural biology to study noncovalent assemblies in native conditions. We report here on a novel method to perform MP analysis in denaturing conditions (dMP) and its application for fast, accurate and straightforward optimization of chemical reactions in cross-linking mass spectrometry (XL-MS) workflows. dMP consists in a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. The proposed single-molecule method clearly overcomes the limitations and outperforms gold standard SDS-PAGE, as illustrated on several biological complexes. dMP provides an unprecedented and unmatched in-solution quantification of all coexisting XL species, including sub-complexes and non-specific XL aggregates, along with identification of significantly higher numbers of XL dipeptides in MS. We anticipate single-molecule dMP to be a high-impact game-changer for the XL-MS community with the potential to leverage the quality and reliability of XL-MS datasets.

biophysics↗

bioRxiv2022

The Ribosome Maturation Factor Rea1 utilizes nucleotide independent and ATP hydrolysis driven Linker remodelling for the removal of ribosome assembly factors

The ribosome maturation factor Rea1 (or Midasin) catalyses the removal of assembly factors from large ribosomal subunit precursors to promote their export from the nucleus to the cytosol. Rea1 consists of nearly 5000 amino-acid residues and belongs to the AAA+ protein family. It consists of a ring of six AAA+ domains from which the {approx} 1700 amino-acid residue linker emerges that is subdivided into stem, middle and top domains. A flexible and unstructured D/E rich region connects the linker top to a MIDAS (metal ion dependent adhesion site) domain, which is able to bind the assembly factor substrates. Despite its key importance for ribosome maturation, the Rea1 mechanism driving assembly factor removal is still poorly understood. Here we demonstrate that the Rea1 linker is essential for assembly factor removal. It rotates and swings towards the AAA+ ring following a complex remodelling scheme involving nucleotide independent as well as nucleotide dependent steps. ATP-hydrolysis is required to engage the linker with the AAA+ ring and ultimately with the AAA+ ring docked MIDAS domain. The interaction between the linker top and the MIDAS domain allows force transmission for assembly factor removal.

biochemistry↗

bioRxiv2020

The importance of charge in perturbing the aromatic glue stabilizing the protein-protein interface of homodimeric tRNA-guanine transglycosylase

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each exchange with respect to dimer and thermal stability or enzyme activity applying native mass spectrometry, thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate strong influence of pH on homodimer stability. Obviously, protonation of a histidine within the aromatic cluster promotes the collapse of an essential structural motif within the dimer interface at slightly acidic pH. TOC GraphicFor table of contents use only. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2020

Molecular determinants of MED1 interaction with the DNA bound VDR-RXR heterodimer

The MED1 subunit of the Mediator complex is an essential coactivator of nuclear receptor-mediated transcriptional activation. While structural requirements for ligand-dependent binding of classical coactivator motifs of MED1 to numerous nuclear receptor ligand-binding domains have been fully elucidated, the recognition of the full-length or truncated coactivator by full nuclear receptor complexes remain unknown. Here we present structural details of the interaction between a large part of MED1 comprising its structured N-terminal and the flexible receptor-interacting domains and the mutual heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) bound to their cognate DNA response element. Using a combination of structural and biophysical methods we show that the ligand-dependent interaction between VDR and the second coactivator motif of MED1 is crucial for complex formation and we identify additional, previously unseen, interaction details. In particular, we identified RXR regions involved in the interaction with structured N-terminal domain of MED1, as well as VDR regions outside the classical coactivator binding cleft affected by coactivator recruitment. These findings highlight important roles of each receptor within the heterodimer in selective recognition of MED1 and contribute to our understanding of the nuclear receptor-coregulator complexes.

molecular biology↗

bioRxiv2020

CRISPR-based bioengineering of the Transferrin Receptor revealed a role for Rab7 in the biosynthetic secretory pathway

The regulated secretory trafficking of neosynthesized transmembrane receptors is particularly challenging to investigate as it is under-represented at steady state compared to the abundance of the other trafficking routes. Here, we combined the retention using selective hook (RUSH) system to a CRISPR/Cas9 gene editing approach (eRUSH) to identify molecular players involved in the trafficking of neosynthesized Transferrin Receptor (TfR) en route to the plasma membrane (PM). TfR-eRUSH monoclonal cells expressing endogenous, ER-retainable and fluorescent TfR were engineered and characterized. Spatiotemporal quantitative proteomics of TfR-eRUSH cells allowed the identification of molecular partners associated with TfR-containing membranes and provided a comprehensive list of potential regulators, co-trafficking cargos, and enriched pathways. Furthermore, we chose to focus our attention on the Rab GTPase family members for their function as vesicle trafficking regulators and performed a Rab-targeted siRNA screen that we correlated to our proteomics data. Unexpectedly, we identified Rab7-harboring vesicles as an intermediate compartment of the Golgi-to-PM transport of the neosynthetic TfR. These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles, also involved in Golgi-to-PM transport. However, Rab6A-TfR vesicles delivered TfR directly to the PM, while in contrast, Rab7A was transiently associated to neosynthetic TfR-containing post-Golgi vesicles but dissociated before PM vesicle fusion. Together, our study proposes the eRUSH as a powerful tool to further study the secretory pathway and reveals an unforeseen role for Rab7 in the neosynthetic transport of the TfR, highlighting the diversity of the secretory vesicles nature for a given cargo.

cell biology↗

bioRxiv2019

PPARG is a tumor suppressor in basal bladder tumors offering new potential therapeutic opportunities.

PPAR{gamma} activation is a critical event in luminal muscle-invasive bladder cancer (MIBC) tumorigenesis, favoring both tumor cell growth and microenvironment modulation toward tumor immune escape. Conversely, the down-regulation of PPAR{gamma} activity in basal MIBC suggests tumor suppressive effects in this subgroup. Here, we report genetic, epigenetic and functional evidence to support the tumor suppressor role for PPAR{gamma} in basal bladder tumors. We identified hemizygous deletions, DNA hyper-methylation and loss-of-function mutations of PPAR{gamma} in basal MIBC, associated with PPAR{gamma} under-expression and its decreased activity. Re-expression of PPAR{gamma} in basal tumor cells resulted in the activation of PPAR{gamma} -dependent transcription program that modulated fatty acid metabolism and cell differentiation and decreased cell growth, which could partly rely on EGFR down-regulation. Structure-function studies of two PPAR{gamma} mutant revealed a destabilization of a region important for coactivator recruitment and should help develop potent molecules to activate PPAR{gamma} as a therapeutic strategy for basal MIBC. The identification of this subtype-dependent dual role of PPAR{gamma} in MIBC strengthens the critical role of PPAR{gamma} in bladder tumorigenesis and reinforces the interest in stratified medicine based on tumor molecular subtyping. One sentence summaryGenetic, epigenetic and functional evidence of a tumor suppressor role for PPAR{gamma} in basal bladder tumors offer new therapeutic opportunities for this subgroup.

cancer biology↗

bioRxiv2019

Aβ(1-42) tetramer and octamer structures reveal edge pores as a mechanism for membrane damage

The formation of amyloid-beta (A{beta}) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimers disease (AD). It is therefore critical to characterize A{beta} oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an A{beta} oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an A{beta}(1-42) tetramer. It comprises a {beta}-sheet core made of six {beta}-strands, connected by only two {beta}-turns. The two faces of the {beta}-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of A{beta}(1-42), we prepared a sample enriched in A{beta}(1-42) octamers, formed by two A{beta}(1-42) tetramers facing each other forming a {beta}-sandwich structure. Notably, samples enriched in A{beta}(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core {beta}-sheets edges of the A{beta}(1-42) tetramers and octamers.

biophysics↗