Biology subjects

Sobota, R.

Publications and source records attributed to Sobota, R..

7 recordsLinked to original sources

bioRxiv2026

Reduced Complex III assembly dampens host inflammation as an adaptive response to viral infection

Mitochondrial electron transport and oxidative respiration are required for immunity and host tolerance. How the ETC contributes to viral infections - where a proinflammatory anti-viral response needs to be finely balanced with host-protective anti-inflammatory mechanisms - remains unclear. Here, we demonstrate that following infection by H1N1 influenza virus, murine macrophages reduce Complex III levels by downregulating the early CIII assembly factor called the COMB complex. To determine the effect of CIII suppression, we utilized Brawnin (Br or UQCC6) knockout mice, which lack COMB complexes and have reduced Complex III. Following H1N1 infection, Br KO bone marrow-derived macrophages (BMDMs) had reduced inflammation due to reduced CIII Qo site ROS. Br KO mice infected with a non-lethal dose of H1N1 had reduced lung immune pathology, viral burden and enhanced recovery following H1N1 infection. Single-cell RNAseq analysis revealed that H1N1-infected Br KO lungs had reduced abundance of hyper-inflammatory monocytes known to cause severe respiratory disease and reduced monocyte chemotaxis. Our study demonstrates that ETC Complex III suppression is part of an innate immune response to viral infection, and is a potential strategy to control host inflammation in acute respiratory viral infections.

immunology↗

bioRxiv2025

A conserved long-range RNA interaction in SARS-CoV-2 recruits ADAR1 to enhance virus proliferation

Long-range RNA-RNA pairing impacts the genome structure and function of SARS-CoV-2 variants. To understand the structure and function relationships of different SARS-CoV-2 variants that have emerged during the COVID-19 pandemic, we performed high-throughput structure probing and modelling of the genomic structures of the wildtype (WT), Alpha, Beta, Delta and Omicron variants of SARS-CoV-2. We observed that genomes of SARS-CoV-2 variants are generally structurally conserved, and that single-nucleotide variations and interactions with RNA binding proteins can impact RNA structures across the viruses. Importantly, using proximity ligation sequencing, we identified many conserved ultra-long-range RNA-RNA interactions, including one that spans more than 17 kb in both the WT virus and the Omicron variant. We showed that mutations that disrupt this 17 kb long-range interaction reduce viral fitness at later stages of its infection cycle, while compensatory mutations partially restore virus fitness. Additionally, we showed that this ultra-long-range RNA-RNA interaction structure binds directly to ADAR1 to alter the RNA editing levels on the viral genome. These studies deepen our understanding of RNA structures in the SARS-CoV-2 genome and their ability to interact with host factors to facilitate virus infectivity.

genomics↗

bioRxiv2024

TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport

Protein aggregation plays key roles in age-related degenerative diseases, but how different proteins coalesce to form inclusions that vary in composition, morphology, molecular dynamics and confer physiological consequences is poorly understood. Here we employed a general reporter based on mutant Hsp104 to identify proteins forming aggregates in human cells under common proteotoxic stress. Over 300 proteins were identified, forming different inclusions containing subsets of aggregating proteins. In particular, TDP43, implicated in Amyotrophic Lateral Sclerosis (ALS), partitions dynamically between two distinct types of aggregates: stress granule and a previously unknown solid inclusion at the ER exit sites (ERES). TDP43-ERES coaggregation is induced by diverse proteotoxic stresses and observed in the motor neurons of ALS patients. Such aggregation causes retention of secretory cargos at ERES and therefore delayed ER-to-Golgi transport, providing a link between TDP43 aggregation and compromised cellular function in ALS patients.

cell biology↗

bioRxiv2021

Identification of an inhibitory pocket in falcilysin bound by chloroquine provides a new avenue for malaria drug development

Despite their widespread use, our understanding of how many antiparasitic drugs work remains limited. We used mass-spectrometry based cellular thermal shift assay (MS-CETSA) to identify possible protein targets of several malaria drugs and drug candidates. We found that falcilysin (FLN) is a common target for several quinoline drugs including chloroquine and mefloquine, as well as drug candidates MK-4815, MMV000848 and MMV665806. At pH 7.5, these compounds all inhibit FLN proteolytic activity with IC50 values ranging from 1.6 to 67.9 {micro}M. Their interaction with FLN was systematically probed by isothermal titration calorimetry and X-ray crystallography, revealing a shared hydrophobic pocket in the catalytic chamber of the enzyme. Characterization of transgenic cell lines with depleted FLN expression demonstrated statistically significant increases in susceptibility towards chloroquine, mefloquine, MK-4815 and MMV000848. Taken together, our findings point to a multimodal mechanism of action for several commonly used anti-malaria drugs. Importantly, a common allosteric pocket of FLN appears amenable to inhibition, providing a structural basis to guide the development of novel drugs against malaria.

microbiology↗

bioRxiv2020

Developmental differentiation in a cytoplasm-dwelling obligate intracellular bacterium

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Its mechanism of cell exit is unusual amongst Rickettsiaceae, as Ot buds off the surface of infected cells enveloped in plasma membrane. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, secreted bacterial effectors, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and macropinocytosis, whereas entry of EB Ot is only sensitive to a macropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen. Author SummaryOrientia tsutsugamushi (Ot) is a bacterial pathogen that causes scrub typhus, a mite-transmitted human disease. This illness is traditionally known to be endemic in the Asia-Pacific, but recent reports of Orientia-like organisms from the Middle East, Africa, and Latin America suggest that it may be globally distributed. Scrub typhus is associated with high mortality if not treated promptly with appropriate antibiotics. Ot is a highly specialized bacterium that can only replicate within living cells, either within the mite vector or in mammalian or human hosts. Ot exits infected cells using a unique mechanism that involves budding off the surface of infected cells. We have discovered that this unusual aspect of its lifecycle involves the bacteria themselves differentiating into a distinct growth form. Different growth forms have not been described in other members of the family Rickettsiaceae, and no other family members have been shown to bud out of host cells in a manner similar to Ot. We find that the two forms of Ot, which we refer to as intracellular and extracellular bacteria (IB and EB respectively), differ in physical properties and protein expression and infect cells through different mechanisms. The identification of structurally and functionally distinct forms of Ot elucidates a vital aspect of this pathogens intracellular life cycle. The two forms are likely to have different antibiotic susceptibilities, therefore our findings may advance the development of novel interventions aimed at inhibiting Ot growth in scrub typhus patients.

microbiology↗

bioRxiv2020

BRAWNIN: A sORF-encoded Peptide Essential for Vertebrate Mitochondrial Complex III Assembly

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution1,2. Here, we show that the mitochondria proteome is enriched for proteins smaller than 100 a.a. (defined as SEPs). Using a mitochondrial prediction and validation pipeline for small open-reading-frame (sORF)-encoded peptides (SEPs), we report the discovery of 16 endogenous mitochondrial SEPs (mito-SEPs) associated with oxidative phosphorylation (OXPHOS). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN (BR), a 71 amino acid peptide encoded by the C12orf73 gene, is essential for respiratory chain complex III (CIII) assembly. In human cells, BR is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In vivo, BR is enriched in muscle tissues and its maternal zygotic deletion in zebrafish causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BR is essential for oxidative phosphorylation across vertebrate species. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

cell biology↗

bioRxiv2019

Dual RNA-seq provides insight into the biology of the neglected intracellular human pathogen Orientia tsutsugamushi

Emerging and neglected diseases pose challenges as their biology is frequently poorly understood, and genetic tools often do not exist to manipulate the responsible pathogen. Organism agnostic sequencing technologies offer a promising approach to understand the molecular processes underlying these diseases. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium and the causative agent of the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning, we investigated the transcriptional architecture of Ot, including operon structure and non-coding RNAs, and found evidence for wide-spread post-transcriptional antisense regulation. We compared the host response to two clinical isolates and identified distinct immune response networks that are up-regulated in response to each strain, leading to predictions of relative virulence which were confirmed in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.

microbiology↗