Biology subjects

Selenko, P.

Publications and source records attributed to Selenko, P..

6 recordsLinked to original sources

bioRxiv2024

α-Synuclein acts as a cholesteryl-ester sensor on lipid droplets regulating organelle size and abundance.

While aggregated alpha-Synuclein (Syn) is commonly associated with Parkinsons disease, its physiological function as a membrane-binding protein is poorly understood. Here, we show that endogenous Syn binds lipid droplets (LDs) in multiple human cell lines and in stem cell-derived dopaminergic neurons. LD-binding encompasses Syn residues 1-100, which masks their detection by immunofluorescence microscopy, probably explaining the scarcity of similar observations in earlier studies. Syn-LD interactions are highly temperature-sensitive and selective for cholesteryl-ester-rich LDs. They promote the formation of Syn multimers that dissociate from LDs at non-permissive temperatures. Syn remains LD-bound throughout starvation-induced lipolysis, whereas siRNA-knockdown diminishes LD abundance and compromises cell viability upon nutrient depletion, without affecting LD biosynthesis. Reciprocally, excess Syn stimulates LD accumulation in dependence of lipid availability, restricts organelle size and ensures intracellular LD organization, which strictly depends on functional membrane-binding. Supporting a general role of Syn in cellular lipid and cholesterol metabolism, our results point to additional loss-of-function similarities between Parkinsons, Alzheimers and Gauchers disease.

cell biology↗

bioRxiv2020

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels.

The Parkinsons disease protein -synuclein (Syn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of Syn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate plasma membrane localization of endogenous pools of Syn in A2780, HeLa, SH-SY5Y and SK-MEL-2 cells. We demonstrate that Syn binds reconstituted PIP2-membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute Syn in cells. We further delineate that Syn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to Syns cellular function(s) at the plasma membrane.

cell biology↗

bioRxiv2020

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics.

Heterogeneous aggregates of the human protein -synuclein (Syn) are abundantly found in Lewy body inclusions of Parkinsons disease patients. While structural information on classical Syn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of Syn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-{beta} amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict Syn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining Syn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative Syn aggregation scavenges cytochrome cs activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits.

biophysics↗

bioRxiv2020

An NMR-based biosensor to measure stereo-specific methionine sulfoxide reductase (MSR) activities in vitro and in vivo.

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments that range from native cell lysates to zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. Our approach further extends high-resolution in-cell NMR measurements of exogenously delivered biomolecules to an entire multicellular organism.

biophysics↗

bioRxiv2019

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging, and chemical biology

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of kinetochores, a spatially confined and well-studied subcellular structures. After electroporation in human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes displayed native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation reconstituted robust kinetochore localization of Spindly. Thus, electroporation is uniquely versatile for delivering synthetic and, as required, chemically modified functional mimics of endogenous proteins, and is therefore a promising tool for chemical and synthetic biology.

cell biology↗

bioRxiv2017

Quantitative mass imaging of single molecules in solution

The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo- and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

biophysics↗