Biology subjects

Sitek, B.

Publications and source records attributed to Sitek, B..

8 recordsLinked to original sources

bioRxiv2026

Quantitative modeling of SARS-CoV-2 replication reveals phase-specific bottlenecks and antiviral targets

SARS-CoV-2 replication depends on a tightly coordinated series of intracellular processes that remain incompletely quantified. Here, we integrated high-resolution time-resolved measurements of viral RNA, protein expression, and infectious virion production with mechanistic mathematical modeling to obtain a quantitative description of the viral replication cycle in human lung cells. Using transcriptomic, proteomic, and infectivity data collected over the first 24 hours of infection, we calibrated an ordinary differential equation model that captures genomic and subgenomic RNA synthesis, viral protein production, virion assembly, and virus release. The model accurately reproduced the observed replication dynamics and enabled estimation of kinetic parameters that are difficult to measure experimentally. Sensitivity analysis identified viral RNA replication and non-structural protein maturation as dominant determinants of viral replication efficiency. To assess predictive power, the model was challenged with independent antiviral perturbation experiments using remdesivir, nirmatrelvir, and montelukast. Model predictions closely matched experimentally observed treatment responses and correctly reproduced drug interaction effects during combination therapy. Furthermore, comparison of alternative mechanistic hypotheses supported NSP5 rather than NSP1 as the primary antiviral target of montelukast. Together, these results establish a predictive framework for dissecting intracellular coronavirus replication and evaluating antiviral intervention strategies.

microbiology↗

bioRxiv2024

Endogenously produced hydrogen cyanide serves as a novel mammalian gasotransmitter

Small, gaseous molecules, known as gasotransmitters (NO, CO, H2S), are produced endogenously in mammalian cells and serve important biological roles. Hydrogen cyanide, traditionally considered a cytotoxic molecule in mammals, serves as an endogenous mediator in several plants and bacterial species. Here we show that low concentrations of cyanide are generated endogenously in mouse liver and human hepatocytes. Cyanide production is stimulated by glycine, occurs at the low pH of lysosomes and requires peroxidase activity. Cyanide, in turn, is detectable in several cellular compartments. Cyanide is also detectable basally in the blood of mice; its levels increase after treatment of the animals with glycine. Rhodanese activity regulates endogenous cyanide levels. Cyanide, when generated endogenously at an optimal level, exerts stimulatory effects on mitochondrial bioenergetics, cell metabolism and cell proliferation. Dysregulation of endogenous cyanide, either below or above optimal levels, impairs cellular bioenergetics. The regulatory effects of cyanide are in part mediated by posttranslational modification of cysteine residues via protein cyanylation; cyanylated protein residues can be detected basally, and increase after treatment with glycine. Controlled low-dose cyanide supplementation exhibits cytoprotective effects, as demonstrated in hypoxia and reoxygenation models in vitro and in vivo. However, pathologically elevated cyanide production, as demonstrated in nonketotic hyperglycinemia - an autosomal recessive disease of glycine metabolism - is deleterious to the cells.

biochemistry↗

bioRxiv2023

The human cytomegalovirus-encoded pUS28 antagonizes CD4+ T-cell recognition by targeting CIITA

Human cytomegalovirus (HCMV) is a relevant pathogen especially for individuals with impaired immunity. Harnessing potent immune antagonists, HCMV circumvents sterile immunity. Given that HCMV prevents the upregulation of human leukocyte antigen (HLA)-DP and HLA-DR, we screened a library of HCMV genes by co-expression with the HLA class II (HLA-II)-inducing transcription coordinator class II transactivator (CIITA). We identified the latency regulator pUS28 as interaction factor and potent viral antagonist of CIITA-driven expression of CD74, HLA-DR, HLA-DM, HLA-DQ, and HLA-DP. Both wt-pUS28 and a mutant incapable to induce G-protein-coupled signaling (R129A), but not a mutant lacking the C-terminus, drastically reduced the CIITA protein abundance post-transcriptionally. While control CD4+ T cells from HCMV-seropositive individuals vigorously responded to CIITA-expressing cells decorated with HCMV antigens, pUS28 expression was sufficient to inhibit HLA-II induction and immune recognition by HCMV-specific CD4+ T cells. Our data uncover a mechanism employed by HCMV to evade HLA-II-mediated recognition by CD4+ T cells.

immunology↗

bioRxiv2022

Epigenetic plasticity via adaptive DNA hypermethylation and clonal expansion underlie resistance to oncogenic pathway inhibition in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. Drug resistance is the major cause for therapeutic failure in PDAC patients with progressive disease. The mechanisms underlying resistance formation are complex and remain poorly understood. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. The effector caspase CASP3 is one of the 114 genes for which transcriptional downregulation inversely correlated with the methylation status of the associated DNA region. CASP3 inactivation in resistant cells led to attenuation of drug-induced apoptosis which could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. Overall, our data provide a context for characterization and targeting of epigenetically mediated resistance mechanisms in PDAC.

cancer biology↗

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

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidAs chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidAs chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N- chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCls chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

biochemistry↗

bioRxiv2021

Differential interferon-α subtype immune signatures suppress SARS-CoV-2 infection

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha () subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFN subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFN subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFN5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFN subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFN5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.

cell biology↗

bioRxiv2019

Relocation of macrophages maintains the barrier function of the urothelium and protects against persistent infection

Macrophages perform essential functions during bacterial infections, such as phagocytosis of pathogens and elimination of neutrophils to reduce spreading of infection, inflammation and tissue damage. The spatial distribution of macrophages is critical to respond to tissue specific adaptations upon infections. Using a novel algorithm for correlative mass spectrometry imaging and state-of-the-art multiplex microscopy, we report here that macrophages within the urinary bladder are positioned in the connective tissue underneath the urothelium. Invading uropathogenic E.coli induced an IL-6-dependent CX3CL1 expression by urothelial cells, facilitating relocation of macrophages from the connective tissue into the urothelium. These cells phagocytosed UPECs and eliminated neutrophils to maintain barrier function of the urothelium, preventing persistent and recurrent urinary tract infection. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY GRAPHICAL ABSTRACT C_FIG_DISPLAY

immunology↗