Biology subjects

Malinovska, L.

Publications and source records attributed to Malinovska, L..

4 recordsLinked to original sources

bioRxiv2026

Deconvolving the structural heterogeneity of alpha-Synuclein in vitro and in situ

The structural states of proteins in cells and tissues provide important insight into their functional states, but studying protein structures in situ remains challenging. Furthermore, a single protein can adopt multiple conformations in cells, which typically cannot be assessed by most structural approaches. Here we developed a novel approach, based on structural proteomics fingerprints, for the quantitative analysis of the distribution of structural states of a protein in vitro and in situ. We applied it to the Parkinsons disease hallmark protein alpha-synuclein (aSyn), for which various structural states (disordered, helical, oligomeric and amyloid fibrillar, among others) have been characterized in vitro, but for which the in vivo structural states remain hotly debated. We measured structure-specific proteolytic fingerprints from well-characterized aSyn in vitro conformations and used them to quantitatively determine the aSyn conformational composition in samples of interest. We first benchmarked our approach using ground truth datasets of known composition and showed that, during in vitro amyloid fibril formation, we could simultaneously detect a time-dependent decrease in disordered monomeric aSyn, an increase in {beta}-sheet-rich oligomers, and a delayed rise in amyloid fibrils. We then applied the method to complex, biologically relevant samples. In a S. cerevisiae aSyn overex-pression model, aSyn was predominantly helical, with an increased helical fraction accompanying its relocalization from the plasma membrane to cytosolic lipid droplets. This shift was linked to proteome-wide changes in lipid droplet homeostasis and fatty acid and ergosterol metabolism, underscoring the role of lipid metabolism and droplet formation in aSyn biology. Importantly, we also detected helical aSyn in human iPSC-derived cortical neurons, supporting the physiological relevance of this conformation. Finally, neurons differentiated from PD patient-derived iPSCs showed elevated levels of {beta}-sheet-rich aSyn compared to wild-type cells. Our approach allowed the in situ identification and quantification of different structural states of aSyn directly in cell lysates. Since several proteins can adopt multiple, functionally-relevant conformations in cells, our approach should be broadly applicable to in situ, quantitative structural and functional studies of proteins.

neuroscience↗

bioRxiv2026

A newly identified three-domain C-type lectin associated with blood feeding in the tick Ixodes ricinus

Ixodes ricinus ticks are widely distributed throughout Europe and represent major vectors of tick-borne encephalitis virus and the Lyme borreliosis agent Borrelia burgdorferi sensu lato. In invertebrates, C-type lectins are commonly associated with innate immune functions, and several such lectins have been predicted in I. ricinus. Given the limited knowledge of lectin function in ticks, we characterized three carbohydrate-recognition domains (CRDs) of a novel C-type lectin identified in the I. ricinus transcriptome (IrCLec). The tertiary structures of CRD1, CRD2, and CRD3, predicted using the AlphaFold 3 program, corresponded to the typical structure of C-type lectins. Conserved carbohydrate-binding motifs were identified in CRD3, whereas non-canonical motifs were present in CRD1 and CRD2. Recombinant His-tagged CRDs were produced and analysed for carbohydrate-binding activity. Glycan array analysis revealed binding of all three domains to selected glycans, while hemagglutination assays demonstrated pronounced binding activity of CRD1 and CRD2 toward human erythrocyte antigens of blood groups A, B, and O. IrCLec expression was highest in the tick midgut and also detected in hemocytes, with expression levels increasing after blood feeding. RNAi-mediated silencing of IrCLec impaired blood feeding efficiency in tick nymphs. Together, these results indicate that IrCLec plays an important role in blood feeding and may additionally participate in lectin-mediated host-pathogen or host-blood component interactions.

biochemistry↗

bioRxiv2025

A flexible end-to-end automated sample preparation workflow enables reproducible large-scale bottom-up proteomics

Bottom-up proteomics holds significant promise for clinical applications due to its high sensitivity and precision, but is limited by labor-intensive, low-throughput sample preparation methods. Advanced automation is essential to enhance throughput, reproducibility, and accuracy and to allow standardization to make bottom-up proteomics amenable for large-scale studies. We developed a fully integrated, automated sample preparation platform that covers the entire process from biological sample input to mass spectrometry-ready peptide output and can be applied on a multitude of biological samples. With this end-to-end solution, we achieved high intra- and inter-plate reproducibility, as well as longitudinal consistency, resulting in precise and reproducible workflows. We showed that our automated workflow surpasses established manual and semi-automated workflows, while improving time efficiency. Finally, we demonstrated the suitability of our automated sample preparation platform for drug development by performing a high-content compound characterization for targeted protein degradation, where high throughput and quantitative accuracy are indispensable. For this, we coupled application-specific workflows to perform proteome profiling and confirm target degradation by precise protein quantification. Overall, our results highlight the selective degradation of specific proteins of interest for ten selected compounds across two cell lines. Thus, the automated sample preparation platform facilitates rapid adaptation to emerging developments in proteomics sample preparation, combining standardization, flexibility, and high-throughput capabilities to drive significant advancements in clinical assays and proteomics research.

biochemistry↗

bioRxiv2023

An approach to characterize mechanisms of action of anti-amyloidogenic compounds in vitro and in situ

Aggregation of amyloidogenic proteins is associated with neurodegenerative disease and its modulation is a focus of drug development efforts. However, the physicochemical properties and structural heterogeneity of amyloidogenic proteins hinder the mechanistic understanding of anti- amyloidogenic compounds. Further, modes of interaction with amyloidogenic proteins are often probed in vitro using purified protein samples, even though these models may not capture in vivo protein structures and do not enable identification of off-target effects. We have developed a modular structural proteomic pipeline based on limited proteolysis coupled to mass spectrometry (LiP-MS) with improved, amino acid level-resolution, to probe the mechanism of action of anti- amyloidogenic compounds. We demonstrate our approach by analysing the interactions of six known or putative anti-amyloidogenic compounds and the amyloid binder Thioflavin T (ThT) with different structural forms of the amyloidogenic Parkinsons disease (PD) protein -Synuclein. Our approach enables determination of putative interaction sites, identification of whether interactions are covalent or non-covalent, and crucially, can probe for interactions of compounds with physiological structures of -Synuclein in complex cell and tissue extracts and identify off-targets. In vitro analyses with our pipeline showed that the green tea polyphenol EGCG induces an N- and C-terminus- dependent compaction of the unstructured -Synuclein monomer, detected preferential interactions of ThT with the N-terminus of -Synuclein fibrils compared to the amyloid core, and showed that the most potent inhibitors of aggregation in our study (EGCG, baicalein and AC Immune compound #2) induced similar non-fibrillar end structures despite different interactions with -Synuclein monomers. Importantly, in mammalian cell lysates, -Synuclein was either a low-affinity target (for EGCG and Baicalein) or did not show evidence of compound interaction (for ThT and doxycycline) in our experimental conditions, despite both monomeric and fibrillar forms interacting with these compounds in vitro. For EGCG, we validated this result in postmortem brain homogenates from PD patients. These in situ analyses identified many additional putative cellular targets of Doxycycline, EGCG, Baicalein and ThT, suggesting that their effects in cellular or animal models of neurodegeneration are likely due to interactions with proteins other than -Synuclein and showing that anti-amyloidogenic compounds should be analyzed in situ as well as in vitro. Our modular pipeline will enable in situ screening of drugs and PET tracers for amyloid aggregates of interest as well as detailed mechanistic studies of compound action in vitro.

systems biology↗