Biology subjects

Steigenberger, B.

Publications and source records attributed to Steigenberger, B..

13 recordsLinked to original sources

bioRxiv2026

DarHTG is a tripartite phage defence system for purine DNA base ADP-ribosylation

Bacteria evolved a variety of phage defence strategies to combat the constant onslaught of viral threats. DarTG toxin-antitoxin systems provide phage defence by ADP-ribosylation of bases in single-stranded DNA which interferes with viral replication and aborts phage infection. Here, we describe DarHTG, a system widespread among bacteria, that combines DarTG with a non-processive superfamily I helicase functioning as co-toxin for DarT and thereby enabling the ADP-ribosylation of double-stranded DNA. We identified two such DarHTG subclasses which catalyse the ADP-ribosylation of adenosine and guanosine bases, respectively, and reverse these modifications with structurally unique ARH superfamily antitoxins. Thus, DarHTG antitoxins expand the known macromolecular target spectrum of the ARH enzyme class to nucleic acids. These DarHTG systems confer resistance to a broad range of bacteriophages and can be triggered by the highly charged tails of viral single-stranded DNA-binding proteins, which occlude the active site of the ARH antitoxin.

microbiology↗

bioRxiv2026

Transmembrane-domain-driven heterodimer assembly licenses integrin head-domain folding in the endoplasmic reticulum

Integrins are a large family of /{beta} heterodimeric receptors whose activation is associated with the extension of the ectodomain and disruption of transient transmembrane domain (TMD) interactions. Whether the TMD interaction serves additional function(s) beyond regulating integrin activation remains unclear. Here, we report that three TMD-disruptive mutations within the integrin {beta}1-subunit activate its function while simultaneously reducing its surface expression. While a Gly744Asn ({beta}1-GN) and Gly744Leu ({beta}1-GL) TMD mutation yielded viable homozygous offspring, the more disruptive Leu748Arg ({beta}1-LR) TMD mutation caused embryonic lethality in mice and drastically reduced {beta}1-integrin surface levels. Cell biology and biochemistry studies revealed that TMD-TMD interactions drive efficient /{beta}-heterodimer assembly, thereby enabling the coupled folding of the ER-luminal {beta}I-domain. Disruption of these TMD interactions leads to endoplasmic reticulum (ER) retention and luminal domain-dependent ER-associated degradation of the monomeric, misfolded {beta}1-subunit. Moreover, mutations that disrupt the /{beta} TMD interaction of multiple integrin classes in humans similarly impair integrin cell surface expression and cause severe loss-of-function diseases. Together, our findings establish that /{beta} TMD association serves a previously unrecognized role in integrin biogenesis by promoting {beta}I-domain folding, preventing premature integrin activation in the ER, and ensuring the proper cell surface expression of activatable integrins.

cell biology↗

bioRxiv2026

The Scm FCS finger binds the Pcl Tudor domain to link canonical PRC1 and PRC2.1 in Drosophila

Canonical Polycomb Repressive Complex 1 (PRC1) was previously reported to selectively associate with Polycomb Repressive Complex 2.1 (PRC2.1) in cross-linking-based affinity purification experiments from Drosophila embryos, but the molecular basis of this interaction has remained unresolved. Here, we established an affinity-purification strategy using cryo-milled Drosophila embryos that recovers intact canonical PRC1 together with the complete PRC2.1 complex without chemical cross-linking. Using AlphaFold3 to systematically model interactions between canonical PRC1 and PRC2.1 subunits, we identified a single high-confidence interface between the FCS finger of the canonical PRC1 accessory subunit Sex comb on midleg (Scm) and the atypical Tudor domain of the PRC2.1 accessory subunit Polycomblike (Pcl). Recombinant Scm FCS finger and Pcl Tudor domain formed a stable complex in vitro, as demonstrated by size-exclusion chromatography and native mass spectrometry. Structure-guided mutagenesis validated the predicted interface: point mutations in either interaction partner abolished complex formation, whereas complementary charge-reversal mutations restored binding. These findings identify the Scm-Pcl interaction as the molecular basis for the selective association between canonical PRC1 and PRC2.1 in Drosophila and reveal that communication between these complexes is mediated by a direct interaction between these accessory subunits.

biochemistry↗

bioRxiv2025

Spatial proteomics reveals mechanisms of cell-intrinsic tryptophan metabolism controlling ovarian cancer survival

Indole-2,3-dioxygenase (IDO1) depletes tryptophan to dampen anti-tumor T cells, yet IDO1 inhibitors (IDO1i) have failed clinically. Using deep visual proteomics, we isolated IDO1 high, medium and low ovarian tumor cells in situ and found IDO1 tightly linked to interferon-{gamma} (IFN-{gamma}) signaling and heterogeneously expressed. Across orthogonal models with tunable IDO1, IFN-{gamma} killed ovarian cancer via a pathway requiring IFN-{gamma} signaling, IDO1-dependent tryptophan depletion, and a biphasic integrated stress response that initially protects from starvation and later drives death. IDO1i or tryptophan supplementation rescued these effects, promoting tumor survival. These data reveal a context-dependent, tumor-suppressive facet of IDO1 and explain how IDO1i can paradoxically favor cancer viability. Our findings call for re-evaluation of IDO1 as a target and suggest exploiting the tryptophan-starvation/GCN2-ISR axis to enhance therapy.

cancer biology↗

bioRxiv2025

IL4i1-sourced oncometabolites promote neuroblastoma cell survival

High-risk neuroblastoma (NB) is driven by the amplification of MYCN in conjunction with additional oncogenic mutations in kinases such as ALK. NB cells require antioxidant responses to maintain redox balance and are highly sensitive to ferroptosis. Here, we show that metabolites derived from infiltrating immune cells expressing IL4i1, a secreted oxidoreductase, are potent suppressors of NB ferroptosis. IL4i1 metabolites (indole-3-pyruvate and 4-hydroxyphenylpyruvate) blocked ferroptosis in all human NB cell lines via a mechanism that depended on free radical scavenging and NRF2 activation but did not require the aryl hydrocarbon receptor. Supernatant transfer experiments confirmed that IL4i1 creates a milieu that protects NB cells from oxidative cell death. Importantly, mice lacking IL4i1 were protected from NB in a high-penetrance MYCN and mutant ALK-driven autochthonous cancer model. Therefore, we propose that immune IL4i1 is permissive for NB growth and survival. IL4i1 produces context-dependent oncometabolites and, as a secreted enzyme, represents a target for cell death manipulation in cancers sensitive to oxidative stress-driven cell death. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/669062v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@6e79baorg.highwire.dtl.DTLVardef@f9a9bcorg.highwire.dtl.DTLVardef@f6b2aaorg.highwire.dtl.DTLVardef@4bb764_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Structural basis of an EMC:Spf1 insertase-dislocase complex in the eukaryotic endoplasmic reticulum

Most eukaryotic membrane proteins are inserted into the membrane at the endoplasmic reticulum (ER). This essential but error-prone process relies on molecular quality control machineries to prevent mistargeting and incorrect structure formation. Here we show that the ER membrane protein complex (EMC) forms an evolutionarily conserved supercomplex with the P5A-ATPase Spf1/ATP13A1. This supercomplex combines the transmembrane domain (TMD) insertase function of the EMC and the TMD dislocase activity of Spf1 in a single entity. Our cryo-EM structure of this supercomplex shows that EMC and Spf1 form a shared intramembrane cavity for substrate engagement and reveals that the ATPase cycle of Spf1 regulates access to this cavity. Together, our study suggests that proteins with opposing biochemical activities in membrane protein biogenesis - insertion versus dislocation - form an integrated molecular machine in eukaryotes to proofread membrane protein insertion and topogenesis.

biochemistry↗

bioRxiv2025

THE IMMUNE RESPONSE AGAINST CANCER IS MODULATED BY STROMAL CELL FIBRONECTIN

Cancer-associated fibroblasts remain poorly understood, with some of them originating from the bone marrow. We therefore took advantage of the diversity of bone marrow stromal cells to shed light on how fibroblasts modulate cancer growth. In two murine cancer models, adding these fibroblasts to tumor cells resulted in smaller lesions. Suppression was enhanced by pretreatment with fibronectin, while genetic deletion of fibronectin in a small subpopulation of stromal cells expressing osterix/sp7 restored growth. The suppressive stromal population showed two more characteristics: the absence of CD31/pecam1 and CD105/endoglin. However, only a decrease in CD105/ENDOGLIN in melanoma patients translated in improved survival. Mechanistically, fibronectin or fibronectin fragments activate integrin 5{beta}1 and TLR4 and increase chemokine production by stromal cells ultimately leading to enhanced recruitment and activity of Ly6G+ myeloid cells without T-cell involvement. This work thus characterizes a beneficial interaction between stromal cells and neutrophils enhancing the immune response against early cancer. HighlightsO_LIStromal cells can be divided into two populations, a tumor supportive one with the potential to stimulate angiogenesis and an inhibitory one with the potential to enhance the immune response C_LIO_LIStromal fibronectin expressing the transduction factor osterix/sp7 produce fibronectin that acts on 5{beta}1 integrin and/or TLR4 on neighboring fibroblasts to modulate Ly6G+ immune cells and suppress tumor growth C_LI One sentence summaryTumor inhibitory fibroblasts have three characteristics and through production of fibronectin act on neighboring cells inducing signaling cascades that ultimately lead to a stronger immune response against early cancer.

cancer biology↗

bioRxiv2025

Modulation of collagen-binding integrins affects fibroblast activation and inhibits fibrosis

The extracellular matrix contributes to the progression of several diseases, sometimes by disrupting organ function such as in lung and liver fibrosis. Because integrin receptors mediate cell-matrix interactions, we conditionally deleted {beta}1 integrin in murine hepatocytes in vivo. Increased TGF-{beta} and matrix deposition ensued. Application of a cyclic peptide (GLQGE) that binds to both 10{beta}1 and 11{beta}1 diminished fibrosis in two murine models. In liver fibrosis, TGF-{beta} production was reduced. In lung fibrosis, however, the effect was exclusively due to suppressing fibroblast activation and hence collagen production without TGF-{beta} involvement. In summary, integrin manipulation successfully changed cell behavior, with effects differing depending on the cell type. Importantly, it is possible to directly suppress fibroblast activation and consequently diminish matrix production independent of disease pathogenesis. This underscores the importance of matrix composition in modifying the behavior of embedded cells and hence disease severity. Key findingsO_LILoss of 11{beta}1 integrin-mediated signal in hepatocytes increases TGF-{beta} and leads to fibrosis C_LIO_LIEngaging 10{beta}1 and/or 11{beta}1 with a cyclic peptide (GLQGE) inhibits fibroblast activation directly and, consequently matrix production and progression of fibrosis C_LI

cell biology↗

bioRxiv2022

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a causal factor in regeneration failure. We demonstrate that the SLRPs Chondroadherin, Fibromodulin, Lumican, and Prolargin are enriched in human, but not zebrafish, CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer structural and mechanical properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as previously unknown inhibitory ECM factors in the human CNS that impair axon regeneration by modifying tissue mechanics and structure. ONE SENTENCE SUMMARYComposition, structural organization, and mechanical properties of the injury ECM direct central nervous system regeneration.

neuroscience↗

bioRxiv2022

Thriving in the heat - Lysine acetylation stabilizes the quaternary structure of a Mega-Dalton hyperthermoactive PEP-synthase

Over time structural adaptations enabled proteins and enzymes to have sufficient stability and flexibility to perform the basic functions of life under various environmental conditions. The catalytic cores of key metabolic enzymes of hyperthermophilic archaea work at a temperature range of 80-120 {degrees}C, similar to the conditions wher the earliest life forms may have thrived. Here we characterize a key enzyme of the central carbon metabolism of Pyrococcus furious, through an integrative approach combining structural mass spectrometry, cryo-electron microscopy, mass photometry and molecular modelling with molecular dynamics simulations. From our investigation, we unveil the structural organization of phosphoenolpyruvate synthase (PPSA). Its 24-meric assembly - weighing over 2 MDa - harbors flexible distal domains, whose proper functioning and coordination depends on widespread chemical acetylation of lysine residues. This non-enzymatic post-translational modification, along with other types of lysine modifications, also occurs on most other major protein complexes of P. furiosus. These modifications likely originated in the chemically favorable primordial conditions and gradually became highly specialized and enzyme-driven in more distantly related mesophiles and Eukaryotes.

biochemistry↗

bioRxiv2021

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection.

Eukaryotic cells employ three SMC complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

biochemistry↗

bioRxiv2020

Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments.

biochemistry↗

bioRxiv2020

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 [A]2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

biochemistry↗