Biology subjects

Spahn, C. M. T.

Publications and source records attributed to Spahn, C. M. T..

10 recordsLinked to original sources

bioRxiv2025

Cryo-EM structure of the naked mole-rat ribosome reveals a stabilized split 28S rRNA

The naked mole-rat (Heterocephalus glaber) is a long-lived mammal with remarkable resistance to cancer and hypoxia, suggesting the evolution of robust proteostasis networks. The ribosome, the central engine of protein synthesis, is key for cellular stress responses and has an unusual feature of the 28S rRNA split; however, the details of its organization remain unknown. Here, we present high-resolution cryo-EM structures of the naked mole-rat 80S ribosome in two states of the elongation cycle. The structures reveal a conserved overall architecture and rRNA modification landscape compared to other mammals, and provide an atomic-level view of the unique break in the 28S rRNA. This cleavage event, located in the D6 expansion segment, is structurally stabilized by a network of interactions with surrounding ribosomal proteins, maintaining the integrity of the large subunit. Our comparative analysis revealed that this compensatory network preserves a canonical architecture nearly indistinguishable from intact mouse and human ribosomes. These findings resolve the structural basis of this unique cleavage, showing that it is a stable, integrated feature whose function is likely linked to more subtle regulatory mechanisms, rather than inducing major structural rearrangements.

biochemistry↗

bioRxiv2025

Membrane protein condensates polymerize actin and form filopodia

Neuronal morphogenesis is guided by filopodia, dynamically generated plasma membrane protrusions filled with parallel actin filaments. However, how filopodial actin filaments are locally produced, organized, and maintained remains unclear. The transmembrane protein PLPPR3 induces filopodia in neurons and other cells. We find that the intracellular domain (ICD) of PLPPR3 forms liquid condensates, which exhibit strong co-partitioning of actin monomers. These condensates promote actin polymerization within the condensates at the expense of actin monomers in the environment, consistent with thermodynamic coupling of actin partitioning and polymerization, which we recapitulate in a modified polymerization kinetics model. This mechanism requires favorable actin partitioning into the condensate relative to the environment. Using crosslinking mass spectrometry, we identify a WH2-like actin-affinity domain within the PLPPR3 ICD. Deleting this domain lowers actin partitioning in vitro and decreases filopodia formation in vivo. Our findings establish a previously unrecognized mechanism for actin network remodeling, in which condensates act as actin sinks, locally boosting monomer concentrations and facilitating polymerization of actin filaments. One sentence summaryOur study uncovers PLPPR3 condensates to locally enrich actin monomers and promote actin polymerization, driving actin network remodeling and neuronal filopodia formation.

cell biology↗

bioRxiv2023

Structural insights into context-dependent inhibitory mechanisms of chloramphenicol in cells

Ribosome-targeting antibiotics represent an important class of antimicrobial drugs. Chloramphenicol (Cm) is a well-studied peptidyl transfer center (PTC) binder, and growing evidence suggests it inhibits translation in a nascent peptide sequence-dependent manner. How such inhibition on the molecular scale translates to action on the cellular level remains unclear. Here, we employ cryo-electron tomography to visualize the impact of Cm inside the bacterium Mycoplasma pneumoniae. By resolving cellular Cm-bound ribosomes to 3.0 [A], we provide atomic detail on Cms coordination and interaction with natural nascent peptides and tRNAs in the PTC. We find that Cm leads to accumulation of translation elongation states that indicate ongoing futile accommodation cycles, and to extensive ribosome collisions. We thus suggest that beyond its inhibition of protein synthesis, the action of Cm may involve activation of cellular stress responses. This work exemplifies how in-cell structural biology advances understanding of mechanisms of action for extensively-studied antibiotics.

biophysics↗

bioRxiv2022

An alpaca-derived nanobody neutralizes the SARS-CoV-2 omicron variant

The SARS-CoV2 Omicron variant sub-lineages spread rapidly through the world, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for anti-SARS-CoV-2 agents that are effective against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production and potential for delivery via inhalation. Here, we characterize the RBD-specific nanobody W25, which we previously isolated from an alpaca, and show superior neutralization activity towards Omicron lineage BA.1 in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike surface glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. Furthermore, we show that W25 also binds the spike protein from the emerging, more infectious Omicron BA.2 lineage with picomolar affinity. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse prioritization of W25 for further clinical development.

microbiology↗

bioRxiv2022

Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism

Human cytomegalovirus (CMV) is a highly relevant and ubiquitously distributed human pathogen. Its rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted the first global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for interferon signalling. Deletion mutagenesis documented that STAT2 is targeted by the viral protein E27. Cellular and in vitro analyses showed that E27 exploits host-derived Cullin4-RING ubiquitin ligases (CRL4) to induce poly-ubiquitylation and proteasomal degradation of STAT2. A cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DDB1- and Cullin4-associated factors (DCAFs) to displace them from the catalytic core of CRL4. Moreover, structural analyses elucidated the mechanism of STAT2 recruitment and indicate that E27-binding additionally disturbs STAT2-dependent interferon signalling by occupying its IRF9 binding interface. For the first time, these data provide structural insights into cytomegalovirus-encoded interferon antagonism and establish an atomic model for STAT2 counteraction by CRL4 misappropriation with important implications for viral immune evasion.

biochemistry↗

bioRxiv2021

Timed global reorganization of protein synthesis during neocortex neurogenesis at codon resolution

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis, and products of translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome number, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing levels of eIF4EBP1, a direct inhibitor of ribosomal protein translation, are concurrent with ribosome downregulation and controls Satb2 fate acquisition during neuronal differentiation. Thus, the refinement of transcriptional programs by translation is central to the molecular logic of brain development. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource: https://shiny.mdc-berlin.de/cortexomics/.

neuroscience↗

bioRxiv2021

Ire1α-Regulated Rate of mRNA Translation is Required for Acquisition of Identity and Polarity in Upper Layer Cortical Neurons

Evolutionary expansion of the neocortex is associated with the increase in upper layer neurons. Here, we present Inositol-Requiring Enzyme 1, Ire1, as an essential determinant of upper layer fate, neuronal polarization and cortical lamination. We demonstrate a non-canonical function of Ire1 in the regulation of global translation rates in the developing neocortex through its dynamic interaction with the ribosome and regulation of eIF4A1 and eEF-2 expression. Inactivation of Ire1 engenders lower protein synthesis rates associated with stalled ribosomes and decreased number of translation start sites. We show unique sensitivity of upper layer fate to translation rates. Whereas eEF-2 is required for cortical lamination, eIF4A1 regulates acquisition of upper layer fate downstream of Ire1 in a mechanism of translational control dependent on 5UTR-embedded structural elements in fate determinant genes. Our data unveil developmental regulation of ribosome dynamics as post-transcriptional mechanisms orchestrating neuronal diversity establishment and assembly of cortical layers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/449563v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@115a09aorg.highwire.dtl.DTLVardef@e30d2dorg.highwire.dtl.DTLVardef@763b27org.highwire.dtl.DTLVardef@2586e9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LISmall molecule screening reveals Ire1 upstream of upper layer neuronal identity C_LIO_LIPolarization and proper lamination of layer II/III neurons require Ire1 C_LIO_LIDevelopment of upper layers requires high translation rates driven by eIF4A1 and eEF-2 downstream of Ire1 C_LIO_LIeIF4A1-dependent Satb2 mRNA translation initiation is a mechanism of upper layer fate acquisition C_LI

neuroscience↗

bioRxiv2021

Structures of active melanocortin-4 receptor--Gs-protein complexes with NDP-α-MSH and setmelanotide

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R- Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP--MSH and setmelanotide, with 2.9 [A] and 2.6 [A] resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP--MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor- Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.

biochemistry↗

bioRxiv2021

Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses

Viruses have evolved means to manipulate the hosts ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins. Author summaryDue to the limited size of virus genomes, virus replication critically relies on host cell components. In addition to the host cells energy metabolism and its DNA replication and protein synthesis apparatus, the protein degradation machinery is an attractive target for viral re-appropriation. Certain viral factors divert the specificity of host ubiquitin ligases to antiviral host factors, in order to mark them for destruction by the proteasome, to lift intracellular barriers to virus replication. Here, we present molecular details of how the simian immunodeficiency virus accessory protein Vpr interacts with a substrate receptor of host Cullin4-RING ubiquitin ligases, and how this interaction redirects the specificity of Cullin4-RING to the antiviral host factor SAMHD1. The studies uncover the mechanism of Vpr-induced SAMHD1 recruitment and subsequent ubiquitylation. Moreover, by comparison to related accessory proteins from other immunodeficiency virus species, we illustrate the surprising variability in the molecular strategies of SAMHD1 counteraction, which these viruses adopted during evolutionary adaptation to their hosts. Lastly, our work also provides deeper insight into the inner workings of the hosts Cullin4-RING ubiquitylation machinery.

biochemistry↗

bioRxiv2020

The architecture of protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

Protein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomes ex vivo from the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, with in vivo embryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/939488v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1231351org.highwire.dtl.DTLVardef@1c940f7org.highwire.dtl.DTLVardef@1c81e2corg.highwire.dtl.DTLVardef@135008b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗