Biology subjects

Schreiber, G.

Publications and source records attributed to Schreiber, G..

At least 19 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↗

bioRxiv2025

Constitutively active RAS prolongs Cdc42 signalling while attenuating MAPK signalling during fission yeast mating

The small GTPase RAS is a signalling hub activating multiple pathways, which may respond differently to a constitutively active RAS mutation. We explored this issue using fission yeast, where RAS-mediated pheromone signalling (PS) activates the MAPKSpk1 and Cdc42 pathways. We observed that in cells with the yeast RAS mutant, ras1.G17V, the MAPKSpk1 activation was attenuated similarly to that in wildtype cells, whereas Cdc42 activation was prolonged. We built a mathematical model implementing PS negative-feedback circuits and competition between the two Ras1 effectors, MAPKKKByr2 and Cdc42-GEFScd1. The model robustly predicted the MAPKSpk1 activation dynamics of an additional 20 PS mutants. In support of the model, a recombinant Cdc42-GEFScd1 competed with MAPKKKByr2 for Ras1 binding and overexpression of the Ras binding domain of either Cdc42-GEFScd1 or MAPKKKByr2 inhibited both downstream pathways. Our study has established that the constitutively active RAS signalling propagates differently to downstream pathways where the system prevents MAPK overactivation.

cell 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↗

bioRxiv2023

RBD-based high affinity ACE2 antagonist limits SARS-CoV-2 replication in upper and lower airways

SARS-CoV-2 has the capacity to evolve mutations to escape vaccine-and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool. Here, we challenged rhesus macaques with SARS-CoV-2 Delta and simultaneously treated them with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment gave equivalent protection in upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 did not block the development of memory responses to Delta and did not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.

immunology↗

bioRxiv2023

Reversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration

For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between pKa and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10-to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated in the cell cytoplasm. Protonation, facilitates the formation of membrane impermeable ionic form of the weak base small molecules. This results in ion trapping, further reducing diffusion rates of weakly basic small molecule drugs under macromolecular crowding conditions where other nonspecific interactions become more relevant and dominant. Our imaging studies showed that acidic organelles, particularly the lysosome, captured these molecules. Surprisingly, blocking lysosomal import only slightly increased diffusion rates and fractional recovery. Conversely, blocking protonation by N-acetylated analogues, greatly enhanced their diffusion and fractional recovery after FRAP. Based on these results, N-acetylation of small molecule drugs may improve the intracellular availability and distribution of weakly basic, small molecule drugs within cells.

biophysics↗

bioRxiv2023

Enhanced transmissibility, infectivity and immune resistance of the SARS-CoV-2 Omicron XBB.1.5 variant

In 2022, we have elucidated the characteristics of a variety of newly emerging SARS-CoV-2 Omicron subvariants. At the end of 2022, the XBB.1.5 variant, an descendant of XBB.1 that acquired the S:F486P substitution, emerged and is rapidly spreading in the USA and is the latest variant of concern. Although the features of XBB.1.5 was already reported by another group as a preprint, we think multiple and independent evaluations important, and these reports are crucial for sustained global health. In this study, our epidemic dynamics analysis revealed that the relative effective reproduction number (Re) of XBB.1.5 is more than 1.2-fold greater than that of the parental XBB.1, and XBB.1.5 is outcompeting BQ.1.1, the predominant lineage in the USA as of December 2022. Our data suggest that XBB.1.5 will rapidly spread worldwide in the near future. Yeast surface display assay and pseudovirus assay respectively showed that the ACE2 binding affinity and infectivity of XBB.1.5 is 4.3-fold and 3.3-fold higher than those of XBB.1, respectively. Moreover, neutralization assay revealed that XBB.1.5 is robustly resistant to BA.2 breakthrough infection sera (41-fold versus B.1.1, 20-fold versus BA.2) and BA.5 breakthrough infection sera (32-fold versus B.1.1, 9.5-fold versus BA.5), respectively. Because the immune resistance of XBB.1.5 is comparable to that of XBB.1, our results suggest that XBB.1.5 is the most successful XBB lineage as of January 2023 by acquiring the S:F486P substitution to augment ACE2 binding affinity without losing remarkable immune resistance, which leads to greater transmissibility.

microbiology↗

bioRxiv2022

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants

In late 2022, the SARS-CoV-2 Omicron subvariants have highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged by recombination of two co-circulating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022 around India. In vitro experiments revealed that XBB is the most profoundly resistant variant to BA.2/5 breakthrough infection sera ever and is more fusogenic than BA.2.75. Notably, the recombination breakpoint is located in the receptor-binding domain of spike, and each region of recombined spike conferred immune evasion and augmented fusogenicity to the XBB spike. Finally, the intrinsic pathogenicity of XBB in hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provided evidence suggesting that XBB is the first documented SARS-CoV-2 variant increasing its fitness through recombination rather than single mutations.

microbiology↗

bioRxiv2022

Convergent evolution of the SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

In late 2022, although the SARS-CoV-2 Omicron subvariants have highly diversified, some lineages have convergently acquired amino acid substitutions at five critical residues in the spike protein. Here, we illuminated the evolutionary rules underlying the convergent evolution of Omicron subvariants and the properties of one of the latest lineages of concern, BQ.1.1. Our phylogenetic and epidemic dynamics analyses suggest that Omicron subvariants independently increased their viral fitness by acquiring the convergent substitutions. Particularly, BQ.1.1, which harbors all five convergent substitutions, shows the highest fitness among the viruses investigated. Neutralization assays show that BQ.1.1 is more resistant to breakthrough BA.2/5 infection sera than BA.5. The BQ.1.1 spike exhibits enhanced binding affinity to human ACE2 receptor and greater fusogenicity than the BA.5 spike. However, the pathogenicity of BQ.1.1 in hamsters is comparable to or even lower than that of BA.5. Our multiscale investigations provide insights into the evolutionary trajectory of Omicron subvariants.

microbiology↗

bioRxiv2022

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques

Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFN2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1-inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN- and IFN-{beta} pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.

immunology↗

bioRxiv2022

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75

SARS-CoV-2 Omicron BA.2.75 emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically different from BA.5, the currently predominant BA.2 descendant. Here, we showed that the effective reproduction number of BA.2.75 is greater than that of BA.5. While the sensitivity of BA.2.75 to vaccination- and BA.1/2 breakthrough infection-induced humoral immunity was comparable to that of BA.2, the immunogenicity of BA.2.75 was different from that of BA.2 and BA.5. Three clinically-available antiviral drugs were effective against BA.2.75. BA.2.75 spike exhibited a profound higher affinity to human ACE2 than BA.2 and BA.5 spikes. The fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were comparable to those of BA.5 but were greater than those of BA.2. Our multiscale investigations suggest that BA.2.75 acquired virological properties independently of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

microbiology↗

bioRxiv2022

Virological characteristics of the novel SARS-CoV-2 Omicron variants including BA.2.12.1, BA.4 and BA.5

After the global spread of SARS-CoV-2 Omicron BA.2 lineage, some BA.2-related variants that acquire mutations in the L452 residue of spike protein, such as BA.2.9.1 and BA.2.13 (L452M), BA.2.12.1 (L452Q), and BA.2.11, BA.4 and BA.5 (L452R), emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these L452R/M/Q-bearing BA.2-related Omicron variants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1 and BA.2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. Furthermore, infection experiments using hamsters indicated that BA.4/5 is more pathogenic than BA.2. Altogether, our multiscale investigations suggest that the risk of L452R/M/Q-bearing BA.2-related Omicron variants, particularly BA.4 and BA.5, to global health is potentially greater than that of original BA.2. HighlightsO_LISpike L452R/Q/M mutations increase the effective reproduction number of BA.2 C_LIO_LIBA.4/5 is resistant to the immunity induced by BA.1 and BA.2 infections C_LIO_LIBA.2.12.1 and BA.4/5 more efficiently spread in human lung cells than BA.2 C_LIO_LIBA.4/5 is more pathogenic than BA.2 in hamsters C_LI

microbiology↗

bioRxiv2022

SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant

Recent studies have revealed the unique virological characteristics of Omicron, the newest SARS-CoV-2 variant of concern, such as pronounced resistance to vaccine-induced neutralizing antibodies, less efficient cleavage of the spike protein, and poor fusogenicity. However, it remains unclear which mutation(s) in the spike protein determine the virological characteristics of Omicron. Here, we show that the representative characteristics of the Omicron spike are determined by its receptor-binding domain. Interestingly, the molecular phylogenetic analysis revealed that the acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidate that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue in another protomer in the spike trimer, which confers the attenuated spike cleavage efficiency and fusogenicity of Omicron. Our data shed light on the evolutionary events underlying Omicron emergence at the molecular level. HighlightsO_LIOmicron spike receptor binding domain determines virological characteristics C_LIO_LISpike S375F mutation results in the poor spike cleavage and fusogenicity in Omicron C_LIO_LIAcquisition of the spike S375F mutation triggered the explosive spread of Omicron C_LIO_LIF375-H505-mediated {pi}-{pi} interaction in the spike determines the phenotype of Omicron C_LI

microbiology↗

bioRxiv2022

Protein Quaternary Structures in Solution are a Mixture of Multiple forms

Over half the proteins form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a proteins oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass-spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle x-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, many proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold Multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins.

biochemistry↗

bioRxiv2022

Genetically engineered MRI-trackable extracellular vesicles as SARS-CoV-2 mimetics for mapping ACE2 binding in vivo

The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex in vivo setup, call for further developments. Here, we show the development of biocompatible genetically engineered extracellular vesicles (EVs) that display the receptor binding domain (RBD) of SARS-CoV-2 on their surface as coronavirus mimetics (EVsRBD). Loading EVsRBD with iron oxide nanoparticles makes them MRI-visible, and thus, allows mapping of the binding of RBD to ACE2 receptors non-invasively in live subjects. Importantly, the proposed mimetics can be easily modified to display the RBD of SARS-CoV-2mutants, namely Delta and Omicron, allowing rapid screening of newly raised variants of the virus. The proposed platform thus shows relevance and cruciality in the examination of quickly evolving pathogenic viruses in an adjustable, fast, and safe manner.

bioengineering↗

bioRxiv2022

Virological characteristics of SARS-CoV-2 BA.2 variant

Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref1, 2), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.

microbiology↗

bioRxiv2021

Ablation of the FACIT collagen XII disturbs musculoskeletal ECM organization and causes patella dislocation and myopathy

Collagen XII, belonging to the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical -chains encoded by the COL12A1 gene. The trimeric molecule consists of three N-terminal noncollagenous NC3 domains joined by disulfide bonds followed by a short interrupted collagen triple helix at the C-terminus. Collagen XII is expressed widely in the musculoskeletal system and mutations in the COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome, which is associated with skeletal abnormalities and muscle weakness. Our study defines the role of collagen XII in patella development using the Col12a1-/- mouse model. Deficiency in Col12a1 expression causes malformed facies patellaris femoris grooves at an early stage, which leads to patella subluxation and growth retardation. Due to the patella subluxation, more muscle fibers with centralized nuclei occur in the quadriceps than in the gastrocnemius muscles indicating a local effect. To further understand the role of collagen XII in the skeletal tissues single cell RNAseq (scRNA-seq) was performed. Comparison of the gene expression in the tenocyte cell sub-population of wild type and Col12a1-/- mice showed that several matrix genes are altered. Finally, we reinvestigated collagen XII deficient patients and observed a patella instability.

genetics↗