Biology subjects

Zoler, E.

Publications and source records attributed to Zoler, E..

5 recordsLinked to original sources

bioRxiv2025

IRF1 Tunes Basal Immunity and Antiviral Readiness in a Context-Dependent Manner

Interferon Regulatory Factor 1 (IRF1) plays a pivotal role in interferon (IFN) signaling, yet its context-dependent regulatory functions remain incompletely understood. Here, we dissect the impact of IRF1 on gene regulation in HeLa cells, by targeted knockout (KO) or overexpression (OE) of IRF1. IRF1 KO did not impair interferon stimulated gene (ISG) expression regulation upon IFN-{beta} stimulation, but partially diminished IFN-{psi} induced gene regulation. IRF1 KO did show a homeostatic role in basal gene abundance, including increasing the abundance of some antiviral genes. RNA-seq analysis showed altered expression of both ISGs and immune signaling genes, implicating IRF1 as a dual regulator that fine-tunes gene abundance through both activation and repression. IRF1 OE induced potent antiviral protection in the absence of exogenous IFN, mediated by type I IFN secretion, particularly of IFN- subtypes. This paracrine effect was confirmed by transcriptomics, cytokine profiling, and mass spectrometry, and was functional even in JAK1-deficient or Ruxolitinib-treated cells but not type I IFN receptor KO cells, suggesting the involvement of non-canonical signaling pathways. Hierarchical clustering of RNA-seq data revealed distinct IFN-independent gene clusters activated or repressed by IRF1, including pathways related to adaptive immunity and T cell function. Using protein-binding microarrays and predictive modeling, we mapped IRF1 binding across promoters and validated functional motifs in the IFIT2 gene promoter by a reporter assay. Our integrative approach establishes IRF1 as a central regulator of antiviral immunity, capable of shaping gene expression both through cytokine signaling and direct promoter binding.

biochemistry↗

bioRxiv2025

Actin Networking Collapse Under STAT5A Deficiency Drives Mitochondrial Dysfunction and Autocrine IFN-beta Production

Coordination between actin cytoskeleton networking and mitochondrial organization and health underpins cellular homeostasis. Here, we found STAT5A to be a pivotal transcription factor that sustains the expression of key actin regulators, including ACTN1. STAT5A, but not STAT5B deficiency dismantles F-actin architecture leading to amorphous cell shape, reduced cellular motility, and corrals mitochondria around the nucleus. This actin networking disruption impairs mitochondrial DRP1 recruitment and dynamic equilibrium, leading to ROS-associated DNA damage and cGAS-STING mediated type I IFN production. Consequently, this establishes a chronically IFN-stimulated state in neighboring cells. Conversely, overexpression of STAT5A increases actin cytoskeleton networking and promotes faster cell motility. Moreover, ectopic expression of ACTN1 in the background of STAT5A-knockout cells is sufficient to restore the actin cytoskeleton organization and mitochondrial network morphology, eliminating DNA damage and IFN-signaling. Giving the importance of actin in cellular homeostasis, our findings place actin abundance, as regulated by the STAT5A-ACTN1 axis as essential for linking cytoskeletal integrity with mitochondrial health, restraining aberrant innate immune activation. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/656095v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1cee57aorg.highwire.dtl.DTLVardef@3be0cdorg.highwire.dtl.DTLVardef@123ed52org.highwire.dtl.DTLVardef@1b1bfcc_HPS_FORMAT_FIGEXP M_FIG C_FIG Key pointsSTAT5A deficiency disrupts cytoskeletal integrity via ACTN1 loss. The disrupted actin-cytoskeleton impairs mitochondria organization, causing ROS production and DNA damage. STAT5A knockout activates cGAS-STING signaling and IFN-{beta} production. Restoring ACTN1 rescues cytoskeleton, mitochondrial function, and immune balance.

cell biology↗

bioRxiv2025

In Vitro and Viral Evolution Convergence Reveal the Selective Pressures Driving Omicron Emergence

In vitro protein evolution provides powerful insights into the amino acid sequences that underlie key biological functions. Here, we used this approach to explore the evolutionary trajectories of the SARS-CoV-2 spike protein receptor-binding domain (RBD) constrained to engage the human ACE2 receptor--an essential first step in viral infection. Applying mild (LSS) or stringent (HSS) selection pressures starting from the ancestral Wuhan strain, we found that HSS, but not LSS rapidly converged on mutations characteristic of the Omicron variant. HSS resulted in fewer, but dominant, non-synonymous mutations mirroring Omicron mutations and its advanced sub-lineages. Conversely, LSS produced only some Omicron-like mutations at much lower frequencies and with incomplete representation. Notably, initiating evolution from Omicron itself resulted in high-fidelity maintenance of Omicron-defining mutations under both HSS and LSS conditions. This evolutionary pattern parallels global SARS-CoV-2 mutation trends as well as in silico simulations, emphasizing the critical role of receptor-binding constraints in shaping viral adaptation, which may be a frequent driver during zoonosis. Predominantly immune evasion associated mutations not selected in vitro. Our findings demonstrate the predictive capacity of in vitro evolution, suggesting Omicrons abrupt emergence resulted from rare, high-stringency selection, superimposed on a background of broader, milder pressures, with Omicron being the humanized SARS-CoV-2.

evolutionary biology↗

bioRxiv2023

Specificity and promiscuity of JAK recruitment regulates pleiotropy of cytokine-receptor signaling

Promiscuous binding of different Janus kinases (JAKs) to class I/II cytokine receptors has been reported, yet its role in signaling is unclear. To systematically explore JAK pairing in type I interferon (IFN-I) signaling, we generated an artificial IFN-I receptor (AIR) by replacing the extracellular domains of IFNAR1 and IFNAR2 with anti mEGFP and mCherry nanobodies. The heterodimeric AIR restored near-native IFN-I activity, while the homomeric variant of IFNAR2 (AIR-dR2) initiated much weaker signaling despite harboring docking sites for signal transducer and activator of transcription (STAT) proteins. AIR-dR1 was signaling inactive, yet, pulldown uncovered its ICD to bind both TYK2 and JAK1. To further investigate the roles of JAKs on the receptors, knockout (KO) JAK1, JAK2, TYK2, and JAK2/TYK2 were generated. JAK1 KO led to complete loss of IFN-I signaling, which was partially restored by TYK2 overexpression. TYK2 KO cells retained partial activity, which was elevated by JAK1 overexpression, suggesting both JAKs to partially substitute each other. Conversely, JAK2 KO only moderately impacted the biological activity of IFN-Is, even in JAK2/TYK2 KO cells. Live cell micropatterning confirmed promiscuous binding of JAK1, JAK2 and TYK2 to IFNAR1 and IFNAR2, in line with an AlphaFold model that shows JAKs interchangeability on IFNAR ICDs. Similar promiscuity of JAK binding was observed for TPOR and GHR but not EPOR, accompanied by different downstream signaling activity. The competitive binding of JAKs to cytokine receptors together with the highly diverse absolute and relative JAK expression levels can account for cell type-dependent signaling pleiotropy observed for cytokine receptors. One Sentence SummaryPromiscuous and interchangeable binding of JAKs to cytokine receptors enables cell type-specific pleiotropic signaling.

biochemistry↗

bioRxiv2021

SARS-CoV-2 RBD in vitro evolution follows contagious mutation spread, yet generates an able infection inhibitor

SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the "European" (20E-EU1), "British" (501.V1),"South African" (501.V2), and Brazilian" variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9[A] Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

biochemistry↗