Biology subjects

Szczepanska, A.

Publications and source records attributed to Szczepanska, A..

6 recordsLinked to original sources

bioRxiv2026

In vivo analysis of UNC-23 reveals residues critical for BAG2 domain function

BAG2 is a co-chaperone that regulates Hsp70 activity and plays key roles in protein homeostasis and disease, yet the residue-level determinants of its stability and function remain poorly defined. Here, we use the Caenorhabditis elegans BAG2 homolog UNC-23 as an in vivo model to dissect these mechanisms. A forward genetic screen identified a missense mutation, UNC-23C403Y, that causes muscle deterioration without affecting developmental timing. Sequence and structural analyses revealed that C403 is highly evolutionarily conserved and located within a structurally conserved BAG domain. Furthermore, to systematically identify residues critical for protein stability, we combined evolutionary conservation with in silico stability predictions and tested selected substitutions in vivo. Despite strong conservation and predicted destabilization, most generated mutations were phenotypically silent, with only UNC-23F416A producing a severe defect, revealing the limited predictive power of current approaches and a high tolerance of the BAG domain to perturbations. Comparative structural analysis further showed that, although the BAG domain architecture is conserved, its core stabilization relies on distinct interactions in invertebrates and vertebrates. Together, our results identify and refine residues contributing to UNC-23/BAG2 function, improving the current understanding of the BAG domain, and demonstrate that its architecture is maintained through distinct stabilizing interactions across evolution.

cell biology↗

bioRxiv2024

Pathogen threat proximity shapes host extracellular vesicle production in pre-infection response

Extracellular vesicles (EVs) play a crucial role in immune responses, yet it remains unclear whether pathogen metabolites alone can stimulate EV production prior to infection. Using Caenorhabditis elegans, we investigate this question through the lens of exophers - large, evolutionarily conserved EVs known to enhance proteostasis, extend lifespan, and improve reproductive fitness. Our study uncovers distinct regulatory mechanisms driving EV production in response to pathogen-derived volatile metabolites and non-volatile secretome, providing insights into host-pathogen signaling before physical interaction. We reveal a sophisticated network that adjusts EV production based on pathogen proximity: non-volatile secretome (including tripeptide Ile-Pro-Pro), signaling an immediate threat, activate immune-dependent EV pathways, while volatile metabolites, forewarning potential danger, initiate immunity-independent exopher production. Both responses rely on sensory neurons, with ASK, ADL, and AWC neurons playing central roles in each, and additional input from ASI, AWB, and ASH neurons specifically enhancing non-volatile secretome-induced EV production. Downstream signal integration is mediated by a circuit involving the RMG hub and AIB/AIA interneurons, with the AIB-specific NPR-9 receptor playing a critical role. Molecularly, we identified multiple GPCRs, including SRI-19, SRI-36/39, and SRR-6, as crucial for the non-volatile response, and revealed that SRR-6 functions in the intestine to regulate muscle exopher production, demonstrating a novel gut-muscle signaling axis. Notably, exposure to volatile pathogen metabolites significantly boosts offspring fitness during infection in SRI-19-dependent manner, albeit at the cost of maternal survival when conditions are being harsher. Thus, our study shows that C. elegans distinguishes pathogen proximity through compound types, activating EV-dependent physiological responses tailored to either immediate or anticipated threats to optimize survival across generations.

physiology↗

bioRxiv2023

Mycelium-bound lipases of psychrophilic and mesophilic fungi as biocatalysts for biodiesel synthesis

In this study, biocatalysts in the form of mycelium-bound lipases of psychrotrophic and mesophilic fungi were screened for their ability to synthesize fatty acid methyl (FAME) and ethyl esters (FAEE). It has been shown that the hydrolytic activity of biocatalysts is not accompanied by their biodiesel synthetic activity. Preliminary studies showed for the first time, that the FTIR technique can be used to predict synthetic activity. The highest correlation between the intensity of the bands and synthetic activity, negative and positive, respectively, was observed for two neighboring bands at 1745 cm-1 (R = -0.73) and 1699 cm-1 (R = 0.68). The psychrotrophic strain Penicillium sp. 59, isolated from the soil of the Arctic tundra (Spitsbergen), has been shown for the first time as an efficient whole-cell biocatalyst for fatty acid ethyl ester synthesis and is stable in organic solvents. Mathematical optimization of biosynthesis was also performed using RSM. A molar canola oil conversion value of 83.3% into FAEE was obtained at 24.2 {degrees}C in n-hexane using 12.5% of used canola oil and 5.3% of the fungal biocatalyst within 6 h. Dry mycelium of Penicillium sp. 59 is a promising new biocatalyst for large-scale biosynthesis of biodiesel, given its low-cost production, high activity at room temperature, no need to mix reactions, and reusability in a minimum of six cycles.

bioengineering↗

bioRxiv2023

New insights into the helicase domain of human Dicer and its biochemical properties: ATPase and RNA rearrangement activities

Vertebrates have one Dicer ortholog that generates both microRNAs (miRNAs) and small interfering RNAs (siRNAs), in contrast to the multiple Dicer-like proteins found in flies and plants. Here, we focus on the functions of the human Dicer (hDicer) helicase domain. The helicase domain of hDicer is known to recognize pre-miRNA substrates through interactions with their apical loop regions. Besides interacting with canonical substrates, the hDicer helicase domain has also been suggested to bind many different cellular RNAs; however, a comprehensive study of the biochemical activities and substrate specificity of the hDicer helicase domain towards different nucleic acids has yet to be undertaken. Here, we conduct such an analysis and reveal that full-length hDicer, through its helicase domain, hydrolyzes ATP. We also show that the hDicer helicase domain binds single-but not double-stranded RNAs and DNAs and that a structural rearrangement of the substrate accompanies the binding of single-stranded RNAs. This RNA rearrangement activity is ATP-independent. Our findings open new avenues for future studies aimed at defining the cellular activities of hDicer that may be associated with these newly described biochemical properties.

molecular biology↗

bioRxiv2022

Pheromone-dependent olfaction bidirectionally regulates muscle extracellular vesicles formation

Extracellular vesicles (EVs) are involved in multiple biological processes; however, there is limited knowledge of the influence of environmental factors or other individuals in a population on EV-regulated systems. The largest evolutionarily conserved EVs, exophers, are a component of the C. elegans maternal somatic tissue resource management system induced by embryos developing in utero, and thus progeny of individuals with active exopher biogenesis (exophergenesis) appear to be privileged. Using this model, we investigated the inter-tissue and social regulatory mechanisms of exophergenesis. We found that the predominant male pheromone, ascr#10, increases exopher production in hermaphrodites via the G-protein-coupled receptor STR-173 in the ASK sensory neurons. In contrast, pheromones from other hermaphrodites in the population temper exophergenesis. Within the hermaphrodite, an increase in embryo accumulation drives pro-exopher signals, and the internal sensory neurons AQR, PQR, and URX play a central role in modulating exopher levels. This intricate process is regulated partly via the neuropeptides FLP-8 and FLP-21, which originate from the URX and AQR/PQR/URX neurons, respectively. Our results reveal a regulatory network integrating internal and external cues, including control of somatic EVs production by the nervous system in response to social signals.

physiology↗

bioRxiv2020

RNA and DNA G-quadruplexes bind to human Dicer and inhibit its activity

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

biochemistry↗