Biology subjects

Serwa, R.

Publications and source records attributed to Serwa, R..

12 recordsLinked to original sources

bioRxiv2026

TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

Chronic myeloid leukemia (CML) is treated with Abl1 tyrosine kinase inhibitors (TKIs). Quiescent cancer cells residing in the bone marrow (BM) can survive the treatment and cause CML relapse. We previously found that a subset of alternative splicing (AS) changes detected in CML cells surviving months of therapy are initiated within hours of treatment onset. Here, we investigated how AS in CML cells is modulated by the human BM microenvironment. By incorporating humanized BM niche models in vivo, we uncovered stroma-induced transcriptome adaptation that influences transcriptional regulation, transmembrane transport, lipid metabolism, the tricarboxylic acid cycle, and respiratory electron transport. We identified RNA-binding protein TIAR (T-cell intracellular antigen-related protein) as a key mediator of CML survival under TKI imatinib treatment. Our data show TIAR-dependent coordination of RNA processing with the metabolic program induced by stromal interaction. Quantitative nascent proteome analysis revealed that TIAR silencing affects the synthesis of metabolic enzymes and proteins involved in imatinib-induced erythroid differentiation. Besides, TIAR knockdown increased lipid peroxidation in untreated cells and decreased reduction potential in cells upon imatinib treatment. Taken together, TIAR deficiency reduces CML survival, possibly by inducing ferroptosis. These findings identify TIAR-dependent RNA processing within the BM niche as a previously unrecognized mechanism of CML therapy resistance and a potential therapeutic vulnerability.

cancer biology↗

bioRxiv2026

Loss of Brg1 promotes seizure development via GABAergic system disruption

The Brahma-related gene 1 (BRG1) encodes the catalytic subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex and plays an important role in brain development. Variants in SWI/SNF components are often found in patients with epilepsy, but it is still unclear how loss of BRG1 function contributes to seizure development. In this study, we analyzed the role of Brg1 in seizure susceptibility using zebrafish models with both pharmacological inhibition or genetic reduction of Brg1. Reduced Brg1 function caused seizure-like behavior and increased neuronal activity in larvae, while basic locomotor activity was preserved. Further analyses showed reduced expression of several GABAergic system markers. In contrast, glutamatergic markers did not show major changes. These results point to a selective impairment of inhibitory signaling. When GABA levels were increased pharmacologically, seizure-like behavior was reduced. This suggests that loss of inhibitory transmission plays an important role in the observed hyperexcitability. Unbiased omics analyses also identified changes in proteins associated with vitamin B6 binding. Treatment with active vitamin B6 reduced seizure-like behavior in larvae with reduced Brg1 function. Taken together, these results indicate that Brg1 is required for proper inhibitory neurotransmission and that partial loss of Brg1 function increases seizure susceptibility. These findings may help to better understand why mutations in chromatin remodeling genes are often associated with epilepsy and could support future studies on targeted modulation of inhibitory signaling in these conditions. Significance StatementChromatin remodeling genes are often mutated in patients with epilepsy, but it is still unclear how these mutations lead to seizures. In this study, we show that reduced function of the chromatin remodeler Brg1 affects inhibitory neurotransmission by impairing the GABAergic system. This leads to increased neuronal activity and seizure-like behavior. Our results identify the chromatin remodeler Brg1 as an important regulator of inhibitory neurotransmission and seizure susceptibility, which may be important for understanding epilepsy associated with neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Lysosomal accumulation of Masitinib alters autophagy via pH-dependent trapping.

Masitinib is an oral tyrosine kinase inhibitor developed initially for the treatment of mastocytosis. Preclinical studies suggest that masitinib can modulate mast cell activity and neuroinflammation, making it a potential therapeutic candidate for neurodegenerative diseases. However, clinical outcomes remain inconclusive, which may be linked to a lack of understanding of its molecular mechanism of action and potential off-target effects. Using a panel of cancer cell lines, we found that masitinib suppresses mTORC1 activity while simultaneously inducing AKT phosphorylation. Interestingly, thermal proteome profiling analysis revealed that lysosomal proteins were the most thermally affected by masitinib. Using large unilamellar vesicle systems that mimic the lipid composition and physicochemical environment of lysosomal membranes, we observed that masitinib preferentially accumulates in acidic vesicle membranes, consistent with a pH-dependent trapping mechanism. Furthermore, cellular assays demonstrated that masitinib disrupts autophagy by impairing lysosomal acidification. Together, these findings elucidate the lysosomal accumulation of masitinib, providing a molecular basis for its mechanism of action.

pharmacology and toxicology↗

bioRxiv2025

SERINC5 Is the Primary Target of Retroviral Antagonists Among Human SERINC Paralogs and Requires a Tri-Cysteine Motif for Its Counteraction by HIV-1 Nef

The human transmembrane protein SERINC5 incorporates into budding HIV-1 particles and inhibits their fusion with new target cells. Among the five members of the SERINC family, SERINC3 and SERINC1 exhibit modest antiviral activity, whereas SERINC2 lacks this function. HIV-1 counteracts SERINC5 by promoting its endocytosis in a Nef- dependent manner. SERINC5 is also downregulated by glycoGag of Moloney murine leukaemia virus (MoMLV) and by the S2 glycoprotein of equine infectious anaemia virus (EIAV). Here, we demonstrate that all human SERINCs, despite their differing antiviral potencies, are incorporated into HIV-1 virions. SERINC5 is the most sensitive to viral antagonists, whereas its paralogs display moderate (SERINC2, SERINC3) or no (SERINC1) susceptibility, suggesting that SERINC5 has exerted stronger selective pressure on retroviral evolution. To identify the determinants of SERINC5 sensitivity to Nef, we performed structure- guided mutagenesis and identified a critical tri-cysteine motif (Cys355, Cys356, Cys358) within intracellular loop 4 (ICL4) required for Nef-mediated downregulation. Substitution of these residues with glutamines conferred resistance to Nef antagonism. While this motif is essential for SERINC5 internalization by diverse HIV-1 and SIV Nef alleles, it is dispensable for downregulation by MoMLV glycoGag and EIAV S2, indicating that these viral antagonists exploit distinct structural features of SERINC5. Finally, we show that SERINC5 is palmitoylated at the tri-cysteine motif, raising the possibility that this modification modulates its sensitivity to Nef. Together, these results confirm the central role of SERINC5 as a target of retroviral counteraction and highlight the crucial importance of ICL4 in mediating its engagement by Nef. ImportanceThe SERINC family of multipass transmembrane proteins includes host factors capable of restricting retrovirus infectivity. Among the five human paralogs, SERINC5 displays the strongest antiviral activity and is the only member efficiently targeted by all known retroviral antagonists, including HIV-1 Nef, MLV glycoGag, and the EIAV S2. This selectivity indicates that the evolutionary pressure exerted by SERINC proteins on retroviruses has been primarily driven by SERINC5 rather than by its relatives. Through structure-guided mutagenesis, we identified a tri-cysteine motif within intracellular loop 4 as a crucial determinant for SERINC5 downregulation by Nef, providing a molecular explanation for its distinctive susceptibility. These findings establish SERINC5 as the principal target of viral counteraction and suggest that its molecular features have contributed to shaping the adaptation of primate lentiviral Nef proteins to the host.

microbiology↗

bioRxiv2025

A dosage-sensitive ALLO-1 network coordinates mitochondrial quality control to enable functional recovery of structurally compromised muscle

Functional recovery of muscle can precede structural repair, yet the mechanisms enabling this dissociation remain elusive. In C. elegans, unc-45(m94) mutants recover motility after heat-induced paralysis despite persisting sarcomeric disorganisation, offering a model to dissect non-contractile drivers of early recovery. Quantitative proteomics identified ALLO-1, a selective autophagy adaptor previously characterised for mediating paternal mitochondrial elimination during embryogenesis, as strongly upregulated during the recovery phase. Proteomic and genetic analyses further revealed a network of ALLO-1-associated factors, including the kinase IKKE-1 (a known ALLO-1 activator), SIP-1 (a small heat shock protein), DIM-1 (a sarcomeric tether), and CAR-1 (an ER-associated RNA-binding protein). In adult muscle, ALLO-1 restrains mitophagy to preserve mitochondrial integrity. Its depletion triggers mitochondrial fragmentation and excessive turnover, especially in the sensitised unc-45(m94) background. Overexpression suppresses mitophagy but reduces oxidative capacity, revealing a dosage-sensitive checkpoint. While IKKE-1 and SIP-1 promote mitochondrial stability, DIM-1 and CAR-1 facilitate turnover when ALLO-1 levels fall below threshold. Together, this regulatory module integrates proteostatic, translational, and mechanical signals to safeguard mitochondrial homeostasis and enable recovery when contractile architecture is compromised.

cell biology↗

bioRxiv2025

Mechanistic Insights into TYSM Protein Regulation by mTORC2 in Response to Chemotherapy.

Transcriptional and translation control of thymidylate synthase (TYMS) is poorly understood, particularly in response to chemotherapeutic drugs such as 5-Fluorouracil (5-FU) and its derivatives. The current study addressed this gap by demonstrating a biphasic response in TYMS protein levels upon 5-FU treatment. Indeed, we observe an initial reduction within the first few hours, followed by a marked increase at 24 hours. These changes occurred independently of transcriptional regulation, as TYMS mRNA levels remained stable during the early phase and showed only a moderate increase later. We further showed that neither autophagy nor proteasomal degradation contributed to this dynamic, but instead it is driven by change in its translation. Using thermal proteome profiling, we identified SIN1, a key component of the mTORC2 complex, as a key regulator of TYMS protein levels. Functional studies revealed that SIN1 depletion negatively alters TYMS levels and dynamics and sensitizes cancer cells to 5-FU-mediated cell death. These findings uncover a novel mechanism controlling TYMS protein levels and suggest that targeting SIN1 may represent a promising strategy to enhance the therapeutic efficacy of 5-FU-based treatments.

biochemistry↗

bioRxiv2025

Temporal and protein-specific S-palmitoylation supports synaptic and neural network plasticity

S-palmitoylation, a dynamic post-translational modification, has long been suggested to play a pivotal role in synaptic plasticity, learning, and memory. However, its precise impact on synaptic proteins and function remains unclear. In this study, we show that acute protein depalmitoylation in the hippocampus differentially affects short- and long-term synaptic plasticity, depending on synapse type. Strikingly, depalmitoylation also reprograms neuronal spiking timing following associative network activation. Our research identifies pre- and postsynaptic proteins dynamically regulated by S-palmitoylation during synaptic plasticity and suggests this modification occurs in isolated excitatory synapses. We also demonstrate that S-palmitoylation targets specific proteins within minutes and is not proteome-wide. These findings mark a significant advance in understanding how lipid modifications drive neural adaptability, memory, and learning.

neuroscience↗

bioRxiv2024

SpoIIDMP-driven peptidoglycan rearrangement is crucial for ribosome translocation into the spore.

In a spore-forming bacterium Bacillus subtilis transcription and translation are uncoupled and the translational machinery is located at the cell poles. During sporulation the cell undergoes morphological changes including asymmetric septation and chromosome translocation. However, the fate of translational machinery during sporulation has not been described. Here, using a combination of microscopic assays and mass spectrometry, we are tracking the ribosome localisation during sporulation in B. subtilis WT and mutants. We show that the ribosomes are associated with the asymmetric septum which is a functionally important organelle and that peptidoglycan rearrangement is essential for ribosome packing into the forespore. We also show that the feeding tube channel SpoIIIA-SpoIIQ is not required for the ribosome translocation, but is essential for maintaining the chromosome inside the spore. One-Sentence SummaryMovement of ribosomes into the spore of B. subtilis follows chromosome transport and is precisely orchestrated in the cell.

microbiology↗

bioRxiv2023

Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation

Translational control during the intricate process of sporulation in Bacillus subtilis as a response to nutrient limitation is still underexplored. Here, we employed a comprehensive approach including RNA-seq, ribosome profiling and fluorescence microscopy to dissect the translational landscape of B. subtilis during sporulation. We identified two events of translation silencing and described the spatiotemporal changes in the subcellular location of translational machinery during sporulation. Using a triple knock-out strain (3KO) of zinc-independents paralogs of three zinc-dependent ribosomal proteins L31, L33 and S14, we investigated the potential regulatory role of ribosome during sporulation. The 3KO strain exhibited delayed sporulation, reduced germination efficiency, and dysregulated translation including expression of key metabolic and sporulation-related genes as well as disruptions in translation silencing, particularly in late sporulation.

microbiology↗

bioRxiv2023

Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptional m6Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

Eukaryotic mRNAs undergo co-transcriptional 5-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am - a common epitranscriptomic mark unique to the mRNA 5-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analog of the m6Am-cap - m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as an RP-HPLC purification handle, allowing separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic setting as anti-cancer vaccines. The biochemical characterization suggests several phenomena underlying the biological properties of AvantCap: (i) increased competitiveness of the mRNA 5-end for eIF4E protein by reducing its propensity for unspecific interactions, (ii) direct involvement of eIF3 in alternative translation initiation, (iii) subtle differences in mRNA impurity profiles, or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of the m6Am in mRNA.

biochemistry↗

bioRxiv2022

Impaired iron recycling from erythrocytes is an early iron-dependent hallmark of aging

Aging affects iron homeostasis, as evidenced by tissue iron loading and toxicity and common anemia in the elderly. Iron needs in mammals are met primarily by iron-recycling from senescent red blood cells (RBCs), a task chiefly accomplished by splenic red pulp macrophages (RPMs) via erythrophagocytosis. Given that RPMs continuously process iron, their cellular functions might be susceptible to age-dependent decline, a condition that has been largely unexplored to date. Here, we found that 10-11-months-old female mice exhibit iron loading, diminished lysosomal activity, and decreased erythrophagocytosis rate in RPMs. These impairments lead to the retention of senescent hemolytic RBCs in the spleen, and the formation of undegradable iron- and heme-rich extracellular protein aggregates, likely derived from ferroptotic RPMs. We further found that feeding mice an iron-reduced diet alleviates iron accumulation in RPMs, enhances their ability to clear erythrocytes, and limits ferroptosis. Consequently, this diet ameliorates hemolysis of splenic RBCs and the formation of iron-rich aggregates, increasing serum iron availability in aging mice. Using RPM-like cells, we show that the diminished iron-recycling capacity of RPMs is underlain by iron accumulation and reduced expression of heme-catabolizing enzyme heme oxygenase 1 (HO-1). Taken together, we identified RPM collapse as an early hallmark of aging and demonstrated that dietary iron reduction improves iron turnover efficacy.

physiology↗

bioRxiv2019

Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we revealing novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA- containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination or primary entering from secondary assembling, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, the work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.\n\nAuthor SummaryBio-orthogonal chemistry and the application of non-canonical amino acid tagging (BONCAT) has opened opportunities for analysis of translational control, protein trafficking and modification in studies of infection and immunity. We expand on our earlier work, reporting the tractable, scalable production and analysis of a large structurally complex enveloped human virus that incorporates non-canonical amino acids into structural proteins from all parts of the virus particle. Thus in the complex translationally altered environment of a herpesvirus infected cell, non-canonical amino acid incorporation has no significant functional effect on the multiple cellular and viral functions required to assemble infectious virions. We further demonstrate the normal recruitment and stochiometries of all detected structural proteins, which combined with data showing unaltered virus yields and infectivity, indicates that the non-canonical residues in the various structural proteins have no effect on their subsequent ability to infect cells. Focusing on spatial analysis of virus entry, we provide examples and reveal novel insight into virus entry and processing. Together with previous reports using bio-orthogonal chemistry in studies of virus infection the potential applications of BMVs are considerable.

microbiology↗