Biology subjects

Marczak, L.

Publications and source records attributed to Marczak, L..

3 recordsLinked to original sources

bioRxiv2026

Loss of a conserved disulfide bond defines Penetration2-related immune myrosinases in Brassicaceae

O_LIThis study investigated whether PEN2/BGLU26 has uniquely evolved as an indole glucosinolate-hydrolysing myrosinase required for Arabidopsis thaliana pre-invasive immunity, or whether related myrosinases can replace its function when targeted to the same subcellular context. C_LIO_LIPEN2-homologous and other selected myrosinases from A. thaliana and Brassica rapa were expressed in the pen2-2 mutant background using a PEN2-like targeting strategy. The resulting lines were assessed by gene expression, protein accumulation, metabolite analysis and pathogen resistance assays. In parallel, targeted mutagenesis, structural comparison and phylogenetic analysis were used to examine molecular and evolutionary features of PEN2-related myrosinases. C_LIO_LIAtBGLU27 and BrBABG.a, but not AtBGLU18, AtBGLU23 or AtBGLU28, partially restored indole glucosinolate hydrolysis and resistance to Colletotrichum tropicale in pen2-2. Unlike AtPEN2, both enzymes acted mainly constitutively and showed distinct substrate preferences. PEN2, BGLU27 and BABG proteins lacked conserved post-translational modification sites, including residues associated with a conserved disulfide bond. Restoring this disulfide bond in AtPEN2 abolished its activity. C_LIO_LIPEN2-related myrosinases form an evolutionarily distinct BGLU lineage associated with indole glucosinolate metabolism in Brassicales. Loss of the conserved disulfide bond appears to be required for PEN2 activity, whereas additional PEN2-specific regulatory features are needed for pathogen-triggered, rather than constitutive, glucosinolate metabolism. C_LI

plant biology↗

bioRxiv2025

Partial Bypass of Frataxin Deficiency by ISCU M141I Restores Cytosolic and Nuclear FeS Cluster Assembly

Iron-sulfur (Fe-S) clusters are essential cofactors required for the activity of numerous proteins involved in fundamental cellular processes, including DNA replication, metabolism and mitochondrial respiration. In eukaryotes, Fe-S cluster biogenesis is initiated in mitochondria by the ISC machinery, which assembles iron and sulfur, delivered by a cysteine desulfurase, onto the scaffold protein ISCU. Frataxin (FXN), a key regulator of this pathway, enhances Fe-S production by accelerating persulfide transfer to ISCU. FXN is essential in eukaryotes, and its loss results in "petite" phenotype in yeast, senescence in dividing mammalian cells and embryonic lethality in mice. Interestingly, in yeast, a methionine to isoleucine substitution at position 141 of the scaffold protein Isu1 can bypass the requirement of FXN. To test whether this bypass mechanism is conserved in mammals, we introduced the equivalent M141I substitution into the endogenous Iscu gene in murine fibroblasts carrying a conditional Fxn allele using CRISPR-Cas9. We show that the ISCU M141I variant enables cell survival in the absence of FXN, preventing cell cycle arrest and decreasing baseline DNA damage. However, these FXN-null survivor clones exhibit slower proliferation, persistent mitochondrial dysfunction and defective mitochondrial Fe-S cluster proteins. In contrast, nuclear and cytosolic Fe-S proteins are preserved, as is cellular iron homeostasis. Importantly, the ISCU M141I variant delays, but does not fully rescue, embryonic lethality in Fxn-deficient mice. Altogether, our results reveal a previously unrecognized compartment-specific rescue of Fe-S cluster dependent processes by the ISCU M141I variant in mammalian cells, raising for the first time the possibility of compartmental regulation of Fe-S cluster biogenesis.

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↗