Biology subjects

Leitner, A.

Publications and source records attributed to Leitner, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A qualitative and quantitative framework for the characterization of protein-RNA interactions by cross-linking mass spectrometry (XL-MS)

Protein-RNA interactions regulate nearly all levels of gene expression. Their characterization at the molecular basis is critical for understanding the function of protein-RNA complexes, both in cell homeostasis and pathology. Yet, molecular interactions underlying the formation of these complexes remain understudied. UV cross-linking mass spectrometry (XL-MS) is one of the leading approaches for in vitro identification of protein-RNA contact sites, and for providing structural information in the form of distance constraints crucial for the refinement of structural models. Our previously published CLIR-MS method enables qualitative characterization of protein-RNA interactions at residue-specific resolution but was not designed for highly accurate quantitative interpretation. Here, we present a XL-MS workflow based on irradiation with UV light that combines data-dependent acquisition (DDA) and parallel reaction monitoring (PRM) for simultaneous detection and characterization of protein-RNA interaction sites at residue-specific resolution, followed by label-free quantification of cross-linked products. We successfully apply our combined workflow to in vitro reconstituted protein-RNA complexes formed by the proteins TDP-43 and FOX1. We report on novel RNA interaction sites located in the oligomerization interface of the N-terminal domain (NTD) of TDP-43, and characterize quantitatively the process of RNA-induced monomerization of the NTD in an RNA titration experiment. We also investigate temperature-dependent differences in the efficiency of cross-link formation in the FOX1 complex. Our framework offers a unique and novel approach to comparative quantification of protein-RNA cross-links, overcoming the limitations imposed by conventional UV XL-MS methods.

biophysics↗

bioRxiv2026

Monomeric TDP-43 binds GU-rich RNA via its NTD but only its oligomers block cryptic exon inclusion

TDP-43 aggregation is a pathological feature of several neurodegenerative diseases, resulting in nuclear loss of function and RNA dysregulation, including cryptic exon derepression. TDP-43 comprises two RNA recognition motifs required for canonical RNA binding, a low-complexity domain implicated in phase separation, and an N-terminal domain (NTD) that mediates oligomerization. While NTD-driven oligomerization is critical for TDP-43 splicing function, some studies suggest a direct interaction of NTD with RNA. Here, we investigated RNA binding by the NTD and its functional interplay with oligomerization. We show that the NTD preferentially binds UG-rich RNA via residues located at the oligomerization interface. The NTD engaged RNA only on monomeric TDP-43, as RNA binding and oligomerization compete for the same interface and rely on similar interaction modes. By disentangling these competing activities, we demonstrated that oligomerization, but not NTD-RNA interaction, is essential for TDP-43-dependent alternative splicing and repression of cryptic exons. Our findings uncover a previously unappreciated RNA-binding activity within the TDP-43 NTD that directly competes with oligomerization and establish oligomerization as a key determinant of TDP-43 cryptic exon repression.

molecular biology↗

bioRxiv2026

Novel protein networks in motile cilia from human epithelial cells revealed by cryo-ET and proteomics analysis of cilia from PCD patients

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

cell biology↗

bioRxiv2026

Neuronal calcium sensors emerge as novel adenylyl cyclase 8 regulators

Adenylyl cyclase 8 (AC8) is a Ca2+-sensitive membrane adenylyl cyclase highly expressed in the central nervous system. AC8-mediated intracellular cyclic adenosine monophosphate (cAMP) accumulation shapes synaptic function, plasticity, and memory formation and is tightly controlled by intracellular Ca2+ and G protein-coupled receptor signaling. Although it is well known that the effects of Ca2+ on AC8 activity are directly mediated by calmodulin, it has remained unclear until now whether other Ca2+-binding proteins also regulate AC8 function. Here, we identify the neuronal calcium sensor hippocalcin-like protein 1 (HPCAL1) as a direct interaction partner of AC8. Using biochemistry, structural proteomics, integrative modeling, cryo-EM and cell-based FRET approaches, we show that HPCAL1 associates with AC8 in a Ca2+-dependent manner. HPCAL1 shows weak positive modulation of AC8 activity in vitro, with sub micromolar affinity. HPCAL1 interacts with flexible regulatory regions of AC8, including the C1b domain and the N- and C-terminal regions. Interestingly, other members of the neuronal calcium sensor family can bind AC8 via the same sites. Together, our study reveals a previously unrecognized mode of Ca2+-dependent AC8 recognition by a group of neuronal calcium sensor proteins, which may be relevant to context-dependent regulation of cAMP signaling.

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↗

bioRxiv2025

Proteomic and structural comparison between cilia from primary ciliary dyskinesia patients with a DNAH5 defect

Primary ciliary dyskinesia (PCD) is a genetic disorder that affects the motile cilia in various organs, leading to recurrent respiratory infections, subfertility, and laterality abnormalities. Traditional diagnostic methods include high-speed video microscopy, immunofluorescence staining, electron microscopy, and genetic screening. However, how different disease-causing variants in the same PCD gene affect clinical presentation, as well as ciliary composition and structure, is not well understood. We investigated the effect of various mutations in DNAH5, an axonemal dynein heavy chain, using mass spectrometry and cryo-electron tomography. We demonstrated differences in the axonemal composition for patients with dnah5 mutations. Furthermore, we showed that reductions in some ciliary components are patient specific. Some of them, KIAA1430, CFAP97, and DTHD1, were not previously recognized as components of human respiratory motile cilia. Finally, we demonstrated that some differences in protein abundance between wild-type and PCD samples can be observed in the 96-nm repeated unit of the axoneme. Our results suggest that many axonemal components are affected in the DNAH5-defective samples. Additionally, our results highlight that disparate mutations have seemingly distinctive consequences for the axonemal composition.

cell biology↗

bioRxiv2024

Interaction between ciliary component proteins from Chlamydomonas revealed by CRISPR/CAS9, cryo-electron tomography and mass spectrometry

To understand molecular mechanism of ciliary beating motion, knowledge of location, interaction and dynamics of >400 component proteins are indispensable. While recent progress of structural biology revealed conformation and localization of >100 proteins, we still need to investigate their networking, art of their interaction and assembly mechanism. We applied CRISPR/CAS9 genome editing technique to the green algae Chlamydomonas to engineer a deletion mutant of a ciliary component, FAP263, located at the distal protrusion, and examined it structurally by cryo-electron tomography (cryo-ET) and mass spectrometry (MS). Cryo-ET and atomic model fitting demonstrated that the FAP263 deletion mutant lacks additional components, FAP78, and FAP184. Unassigned density near FAP263 in the cryo-ET map of WT cilia is likely FAP151, as suggested by cross-linking mass spectrometry. Based on the structure, we modeled how these four proteins might form a complex. Furthermore, it was shown that dynein f phosphorylation is inhibited in the FAP263 mutant, indicating an important role of this protein complex for dynein f phosphorylation. Our study demonstrates a novel approach to investigate protein networking inside cilia.

cell biology↗

bioRxiv2023

Tissue Transglutaminase 2 has higher affinity for relaxed than for stretched fibronectin fibers

Tissue transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN-fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal {beta}-sandwich and C-terminal {beta}-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2s C-terminal {beta}-barrels that interacts with FN regions outside of the canonical gelatin binding domain, specifically FNI2 and FNIII14-15. Combining XL-MS distance restraints with molecular docking reveals the mechano-regulated binding mechanism between TG2 and modules FNI7-9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how force regulate ECM deposition and maturation, and how TG2 mediates cell-ECM adhesion in health and in various pathophysiological processes. Data are available via ProteomeXchange with identifier PXD043976.

molecular biology↗

bioRxiv2023

Cross-linking mass spectrometric analysis of the endogenous TREX complex from S. cerevisiae

The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3 end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In S. cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1 and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer. The overall structures of the recombinant yeast THO-Sub2 complexes strongly resemble the structural conformation of the monomers and the dimer interface of the endogenous TREX complex.

biochemistry↗

bioRxiv2023

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate the folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. In the open TRiC state, PhLP2A binds to the chambers equator while its N-terminal H3-domain binds to the apical domains of CCT3/4, thereby displacing PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to the positively charged inner surfaces formed by CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding. Highlights- Structural analysis of TRiC-mediated folding cycle with cochaperones PhLP2A and PFD. - The interactions of PhLP2A and PFD with TRiC are mutually exclusive. - PhLP2A domains interact in a subunit-specific manner with the TRiC chamber. - PhLP2A domains are rearranged in ATP-closed TRiC to contact actin across the ring interface

biophysics↗

bioRxiv2023

Structural basis of activation and inhibition of the Ca2+/calmodulin-sensitive adenylyl cyclase 8

Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, including cognitive functions and memory, the structural basis of its regulation by G proteins and CaM is not well defined. Here we report the 3.5 [A] resolution cryo-EM structure of the bovine AC8 bound to Ca2+/CaM and the stimulatory Gs protein. The structure reveals the architecture of the ordered AC8 domains bound to Gs and a small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry, allow us to identify the contact sites between AC8 and its regulators, CaM, Gs, and G{beta}{gamma}, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

biochemistry↗

bioRxiv2022

ROS induction as a strategy to target persister cancer cells with low metabolic activity in NRAS mutated melanoma

Metabolic reprogramming is an emerging hallmark of resistance to cancer therapy but may generate vulnerabilities that can be targeted with small molecules. Multi-omics analysis revealed that NRAS-mutated melanoma cells with a mesenchymal transcriptional profile adopt a quiescent metabolic program to resist cellular stress response induced by MEK-inhibitor resistance. However, as a result of elevated baseline ROS levels, these cells become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intra-tumor heterogeneity requires the combination of a ROS-inducer and a MEK-inhibitor to target both tumor growth and metastasis. By ex vivo pharmacoscopy of 62 human metastatic melanomas, we found that MEK-inhibitor resistant tumors significantly benefitted from the combination therapy. Finally, we profiled 486 cancer cell lines and revealed that oxidative stress responses and translational suppression are biomarkers of ROS-inducer sensitivity, independent of cancer indication. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS-inducers in melanoma and other cancers. Statement of SignificanceTargeted-therapy resistance in cancer arises from genetic selection and both transcriptional and metabolic adaptation. We show that metabolic reprogramming sensitizes resistant cells to ROS-induction in combination with pathway inhibitors. Predictive biomarkers of metabolic sensitivity to ROS-inducing agents were identified in many cancer entities, highlighting the generalizability of this treatment approach. Graphical summary O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cancer biology↗

bioRxiv2022

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/507991v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@183d480org.highwire.dtl.DTLVardef@1f4235aorg.highwire.dtl.DTLVardef@13d1aedorg.highwire.dtl.DTLVardef@b2c87f_HPS_FORMAT_FIGEXP M_FIG C_FIG The ongoing pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further drug development.

biophysics↗

bioRxiv2022

Integrative solution structure of a PTBP1-viral IRES complex reveals strong compaction and ordering with residual conformational flexibility

RNA-binding proteins (RBPs) are crucial regulators of gene expression and often comprise well-defined domains interspersed by flexible, intrinsically disordered regions. The structure determination of ribonucleoprotein complexes involving such RBPs is not common practice and requires integrative structural modeling approaches due to the fact that they often do not form a single stable globular state. Here, we integrate data from magnetic resonance, mass spectrometry, and small angle scattering to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1 also called hnRNP I) bound to an RNA which is part of the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). PTBP1 binding to this IRES element enhances translation of the viral RNA. The determined structural ensemble reveals that both RNA and protein experience a strong compaction upon complex formation, get ordered but still maintain a substantial conformational flexibility. The C-terminal RNA recognition motif (RRM4) of PTBP1 rigidifies the complex by binding a single-strand RNA linker and, in turn, is essential for IRES-mediated translation. PTBP1 acts as an RNA chaperone for the IRES, by ordering the RNA into a few discrete conformations that expose the RNA stems to the outer surface of the RNP complex for subsequent interactions with the translation machinery. The conformational diversity within this structural ensemble is likely common among RNP complexes and important for their functionality. The presented approach opens the possibility to characterize heterogeneous RNP structures at atomic level.

biochemistry↗

bioRxiv2022

Sensitive detection and structural characterisation of UV-induced cross-links in protein-RNA complexes using CLIR-MS

Protein-RNA interactions underpin many critical biological processes, demanding the development of technologies to precisely characterize their nature and functions. Many such technologies depend upon cross-linking under mild irradiation conditions to stabilize contacts between amino acids and nucleobases; for example, the cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) method. A deeper understanding of the CLIR-MS workflow is required to maximize its impact for structural biology, particularly addressing the low abundance of cross-linking products and the information content of spatial/geometric restraints reflected by a cross-link. Here, we present a vastly improved CLIR-MS pipeline that features enhanced sample preparation, data acquisition and interpretation. These advances significantly increase the number of detected cross-link products per sample. We demonstrate that the procedure is robust against variation of key experimental parameters, including irradiation energy and temperature. Using this improved protocol on four protein-RNA complexes representing canonical and non-canonical RNA-binding domains, we propose for the first time the distances encoded by protein-RNA cross-links, enabling their use as structural restraints. We also compared the cross-linking of canonical RNA with 4-thiouracil-labeled counterparts, showing slight, but noticeable differences. The improved understanding of protein-RNA cross-links refines the structural interpretation of complexes and facilitates the adoption of the method in integrative/hybrid structural biology.

biochemistry↗

bioRxiv2022

Structural visualization of the tubulin folding pathway directed by eukaryotic chaperonin TRiC

SUMMARYThe ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of {beta}-tubulin using human Prefoldin and TRiC. We find unstructured {beta}-tubulin is delivered by Prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. CryoEM resolves four near-atomic resolution structures containing progressively folded {beta}-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N-domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Disordered CCT C-termini within the chamber promote subsequent folding of tubulin Core and Middle domains and GTP-binding. Thus, TRiCs chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

biochemistry↗

bioRxiv2021

Towards a structurally resolved human protein interaction network

All cellular functions are governed by complex molecular machines that assemble through protein-protein interactions. Their atomic details are critical to the study of their molecular mechanisms but fewer than 5% of hundreds of thousands of human interactions have been structurally characterized. Here, we test the potential and limitations of recent progress in deep-learning methods using AlphaFold2 to predict structures for 65,484 human interactions. We show that higher confidence models are enriched in interactions supported by affinity or structure-based methods and can be orthogonally confirmed by spatial constraints defined by cross-link data. We identify 3,137 high confidence models, of which 1,371 have no homology to a known structure, from which we identify interface residues harbouring disease mutations, suggesting potential mechanisms for pathogenic variants. We find groups of interface phosphorylation sites that show patterns of co-regulation across conditions, suggestive of coordinated tuning of multiple interactions as signalling responses. Finally, we provide examples of how the predicted binary complexes can be used to build larger assemblies. Accurate prediction of protein complexes promises to greatly expand our understanding of the atomic details of human cell biology in health and disease.

bioinformatics↗

bioRxiv2021

Molecular architecture of the human tRNA ligase complex

RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.

biochemistry↗