Biology subjects

Figiel, M.

Publications and source records attributed to Figiel, M..

9 recordsLinked to original sources

bioRxiv2026

Structures and enzymatic mechanisms of DRT7/UG10 antiphage reverse transcriptases

The ongoing evolutionary arms race between bacteria and bacteriophages has driven the emergence of numerous anti-phage defense systems. Several of these defenses contain domains related to reverse transcriptases (RTs), DNA polymerases that utilize RNA as a template. Certain members of the Abi/UG RT family employ a unique enzymatic mechanism combining protein-primed DNA synthesis with template-independent polymerization. A subgroup, UG10 - also known as Defense-related RT 7 (DRT7) - is characterized by an enzymatic core comprising an RT-like domain and a primase domain, accompanied by a poorly characterized accessory element. This element is either C-terminally fused to the core or encoded by a separate open reading frame. Here, we present cryo-EM structures of protein-DNA conjugates from two UG10 enzymes, revealing a compact yet flexible domain architecture. We demonstrate that the poly(dT) product synthesized by the RT-like domain serves as a template for the primase domain to generate a complementary poly(A) strand. Combining biochemical experiments with structural modeling, we propose the conformational changes necessary for protein priming and coordination between the RT and primase domains. Finally, we show that DRT7 exhibits broad-spectrum anti-phage activity and demonstrate that the {lambda} phage DNA mimic protein Gam can trigger this defense system.

molecular biology↗

bioRxiv2025

Structural insights into DNA annealing and recombination by herpesviral DNA-binding proteins ICP8 and BALF2

The DNA-binding proteins of herpesviruses are key factors involved in multiple stages of DNA replication and recombination. The well-studied ICP8 protein of Herpes Simplex Virus 1 binds single-stranded DNA in a cooperative manner and facilitates the annealing of complementary DNA strands in a process that involves the formation of double helical filaments. Moreover, it cooperates with UL12 alkaline nuclease to execute recombination between homologous DNA sequences. In this work, we present the structures of ICP8 and its homolog BALF2 of the Epstein-Barr Virus in complexes with DNA. These structures reveal a conserved conformation of the bound DNA in which several bases are rotated away from the protein, enabling homology search and base-pairing. The crystal structures of ICP8-DNA depict the pairing between two DNA strands, showing the flexibility of base-pairing. In the structure of the double filament of ICP8, we visualize the interactions between protein subunits within and between the antiparallel strands. Finally, we present the first complete structure of a ternary recombinase complex composed of ICP8 and UL12 bound to a model DNA. This structural landscape illustrates the different stages of the DNA recombination process in herpesviruses, shedding light on molecular mechanisms of their replication.

molecular biology↗

bioRxiv2024

Identification of neurodevelopmental organization of the cell populations of juvenile Huntington's disease using dorso-ventral HD organoids and HD mouse embryos

Huntingtons disease (HD), especially juvenile-onset HD (JOHD), involves early neurodevelopmental pathogenesis alongside the gradual breakdown of the corticostriatal neural axis. To better understand this mechanism, we created fused dorsal-ventral forebrain organoids from induced pluripotent stem cells (iPSCs) from JOHD to mimic early corticostriatal interactions in the disease. We observed characteristic growth phenotypes in HD organoids and found that these phenotypes were influenced by paracrine signals from opposite regions (dorsal-ventral and ventral-dorsal). These phenotypes were only partially rescued by conditioning with medium in HD compared to control organoids. We also investigated humanized HD embryonic mouse forebrains at E13.5 to validate the phenotypes. Using single-cell RNA sequencing (scRNAseq) and immunofluorescence of the internal structure of HD organoids, we observed early neurodevelopmental signs, including stalling during the phase of increased progenitor cell growth, delayed neuron maturation, and disrupted patterning between pallial and subpallial regions. A consistent feature across in vitro and in vivo models was an abnormal expansion of transthyretin (TTR)-positive cells resembling choroid plexus (ChP), along with ectopic expression of neuronal factors in ChP and a reduction in populations expressing intermediate progenitor and interneuron markers. In mosaic organoids that combined healthy and JOHD tissues, the healthy environment helped reduce ChP overgrowth and restore normal progenitor and neuronal development. This suggests that some developmental defects caused by mutant HTT are reversible and influenced by non-cell-autonomous factors. Overall, these findings point to early ChP-related abnormalities as a key aspect of JOHD neurodevelopmental disturbance and propose the ChP-CSF environment as an important area for future mechanistic studies and potential therapies.

developmental biology↗

bioRxiv2023

The Cell Subtypes Selection by Genes (CSSG) algorithm for discovering cell populations in high resolution

The recent massive improvements in transcriptomics and single-cell technologies have led to a rising volume of data and demand for advances in bioinformatics processing. Existing methods are not fully capable of discovering genetic markers responsible for high-resolution cellular tissue heterogeneity, cell lineages during organism development, and cell differentiation with rare intermediate populations. In response to demand, we have generated a new Cell Subtypes Selection by Genes (CSSG) algorithm which is supported by a dedicated and fully automatic JSEQ(R) pipeline. The new CSSG algorithm is iterative, parallel, and able to make decisions for discovering cell populations in tissues based on transcript occurrence in cells. The CSSG/JSEQ is complemented by a new strategy and specialized algorithm for the naming of cell populations. Our approach allows for high-resolution tracing of cell populations, finding relations and hierarchy between them, particularly important for complex tissues such as the brain. The pipeline allows the establishment of developmental, differentiation, and pathogenic trajectory and takes a "snapshot" of a current physiological or pathological cellular stage of the investigated organ at the transcriptional level.

bioinformatics↗

bioRxiv2022

PKB/AKT attenuates Lewy Body-like pathology in primary neurons via Cathepsins B and D

Motor symptoms of Parkinsons disease (PD) are caused by the loss of dopamine neurons in the substantia nigra pars compacta. Current PD therapies offer only symptomatic relief, and the research on novel ways to stop or slow down the degeneration of dopamine neurons in PD attracts considerable efforts. A characteristic feature of PD is the abnormal accumulation and spread of misfolded -synuclein (-syn) protein leading to the formation of Lewy neurites and bodies in different neuronal populations, including dopamine neurons. The process of Lewy body formation compromises neuronal functions and contributes to the progression of the disease and neurodegeneration in PD patients. Treatments preventing -syn misfolding, aggregation and/or spread may therefore provide much-needed disease-modifying therapies for PD. We and others have demonstrated that -syn aggregation and Lewy body formation can be modelled in primary neuronal cultures treated with -syn pre-formed fibrils (PFFs). Using this approach, we have identified several factors preventing the accumulation of phosphorylated -syn in dopamine neurons in vitro and in vivo. In particular, we and others have demonstrated a prominent role of PKB/AKT pathway activation in promoting the survival of dopamine neurons. Here, we utilize lentiviral vectors (LVs) to express constitutively active myristoylated serine-threonine protein kinase AKT1 (mAKT1) in primary dopamine neurons. We show that lentivirally-delivered mAKT1 promoted survival of dopamine neurons after thapsigargin-induced endoplasmic reticulum (ER) stress and prevented fibril-induced accumulation of phosphorylated -syn. Furthermore, using pharmacological tools, we demonstrate that this activity of mAKT1 is dependent on the functional Cathepsins B and D. These proof-of-principle results demonstrate the critical importance of lysosomal enzymes for processing misfolded -syn and establish a basis for the use of LVs to develop neuroprotective gene therapy strategies for dopamine neurons in PD.

neuroscience↗

bioRxiv2021

The DMT and Psilocin Treatment Changes CD11b+ Activated Microglia Immunological Phenotype

Psychedelics are new, promising candidate molecules for clinical use in psychiatric disorders such as Treatment-Resistant Depression (TRD) and Post Traumatic Stress Disorder (PTSD). They were recently also proposed as molecules supporting neural tissue repair by anti-inflammatory properties. Here we reported that two classic psychedelics, DMT and psilocin, can influence microglial functions by reducing the level of TLR4, p65, CD80 proteins, which are markers of the immune response, and upregulat TREM2 neuroprotective receptor. Psilocin also secured neuronal survival in the neuron-microglia co-culture model by attenuating the phagocytic function of microglia. We conclude that DMT and psilocin regulate the immunomodulatory potential of microglia. Of note, psychedelics were previously reported as a relatively safe treatment approach. The demonstrated regulation of inflammatory molecules and microglia phagocytosis suggests that psychedelics or their analogs are candidates in the therapy of neurological disorders where microglia and inflammation significantly contribute to pathogenic disease mechanisms.

cell biology↗

bioRxiv2021

Broad influence of mutant ataxin-3 on the proteome of the adult brain, young neurons, and axons reveals central molecular processes and biomarkers in SCA3/MJD using knock-in mouse model

Spinocerebellar ataxia type 3 (SCA3/MJD) has a polyQ etiology but, the current knowledge on molecular processes and proteins involved in pathogenesis is not sufficient to fully determine the disease mechanism and find drug targets. Proteases and deubiquitinases, such as Ataxin-3, often have a profound impact on other proteins, yet the global model picture of SCA3 disease progression on the protein level, showing the most crucial proteins and pathways in the brain, and neurons, was not investigated previously. Here, we investigated molecular SCA3 mechanism using interdisciplinary research paradigm combining SCA3 knock-in model, behavior, MRI, brain proteomics, precise axonal proteomics, neuronal energy recordings, labeling of vesicles, and inclusions and focusing in axonal compartment. We have demonstrated that altered metabolic and mitochondrial proteins in the brain and the lack of weight gain in Ki91 SCA3/MJD mice is reflected by the failure of energy metabolism recorded in neonatal SCA3 cerebellar neurons. We have determined that further, during disease progression, proteins responsible for metabolism, cytoskeletal architecture, vesicular and axonal transport proteins are disturbed, revealing axons as one of the essential cell compartments in SCA3 pathogenesis. Therefore we focus on SCA3 pathogenesis in axonal and somatodendritic compartments revealing highly increased axonal localization of protein synthesis machinery, including ribosomes, translation factors, and RNA binding proteins, while the level of proteins responsible for cellular transport, and mitochondria was decreased. We demonstrate the accumulation of axonal vesicles in neonatal SCA3 cerebellar neurons and increased phosphorylation of SMI-312 positive adult cerebellar axons, which indicate axonal dysfunction in SCA3. In summary, the SCA3 disease mechanism is based on the broad influence of mutant ataxin-3 on the neuronal proteome. Processes central in our SCA3 model include disturbed localization of proteins between axonal and somatodendritic compartment, early neuronal energy deficit, altered neuronal cytoskeletal structure, an overabundance of protein synthetic machinery in axons.

neuroscience↗

bioRxiv2021

Juvenile Huntington's Disease and Other PolyQ diseases, Update on Neurodevelopmental Character and Comparative Bioinformatic Review of Transcriptomic Data

Polyglutamine (PolyQ) diseases are neurodegenerative disorders caused by the CAG repeat expansion mutation in affected genes resulting in toxic proteins containing a long chain of glutamines. There are nine PolyQ diseases: Huntingtons disease (HD), spinocerebellar ataxias (types 1, 2, 3, 6, 7, and 17), dentatorubral-pallidoluysian atrophy (DRPLA), and spinal bulbar muscular atrophy (SBMA). In general, longer CAG expansions and longer glutamine tracts lead to earlier disease presentations in PolyQ patients. Rarely, cases of extremely long expansions are identified for PolyQ diseases, and they consistently lead to juvenile or sometimes very severe infantile-onset polyQ syndromes. In apparent contrast to the very long CAG tracts, shorter CAGs and PolyQs in proteins seems to be the evolutionary factor enhancing human cognition. Therefore, polyQ tracts in proteins can be modifiers of brain development and disease drivers, which contribute neurodevelopmental phenotypes in juvenile- and adult-onset PolyQ diseases. Therefore we performed a bioinformatics review of published RNAseq polyQ expression data resulting from the presence of polyQ genes in search of neurodevelopmental expression patterns and comparison between diseases. The expression data were collected from cell types reflecting stages of development such as iPSC, neuronal stem cell, neurons, but also the adult patients and models for PolyQ disease. Our comparative bioinformatic review highlighted several (neuro)developmental pathways and genes identified within PolyQ diseases and mouse models responsible for neural growth, synaptogenesis, and synaptic plasticity.

neuroscience↗

bioRxiv2021

Surviving hypothermia by ferritin-mediated iron detoxification

How animals rewire cellular programs to survive cold is a fascinating problem with potential biomedical implications, ranging from emergency medicine to space travel. Studying a hibernation-like response in the free-living nematode Caenorhabditis elegans, we uncovered a regulatory axis that enhances the natural resistance of nematodes to severe cold. This axis involves conserved transcription factors, DAF-16/FoxO and PQM-1, which jointly promote cold survival by upregulating FTN-1, a protein related to mammalian Fth1/ferritin. Moreover, we show that inducing expression of Fth1 also promotes cold survival of mammalian neurons, a cell type particularly sensitive to deterioration in hypothermia. Our findings in both animals and cells suggest that FTN-1/Fth1 facilitates cold survival by detoxifying ROS-generating iron species. We finally show that mimicking the effects of FTN-1/Fth1 with drugs protects neurons from cold-induced degeneration, opening a potential avenue to improved treatments of hypothermia.

physiology↗