Biology subjects

Novakovic, M.

Publications and source records attributed to Novakovic, M..

5 recordsLinked to original sources

bioRxiv2025

Multiplexed analysis of multicomponent biomolecular condensates without any tag

Biomolecular condensates, formed in the process of liquid-liquid phase separation (LLPS), play key roles in RNA metabolism and cellular organization. These dynamic assemblies often contain several components such as proteins and RNAs. Experimental methods to study biological condensates commonly involve fluorophore-labelling of the biomolecules, which may affect phase separation behavior and in addition cannot access dilute-phase components due to low dye concentrations. Here we introduce a non-invasive, multiplexed NMR approach that enables selective observation of multiple components in hydrogel-stabilized biphasic condensates without using tags. The introduced multiplexing filter combines enhanced spin-spin cross-relaxation in the condensed phase with diffusion and isotope filters to resolve signals from distinct protein pools from both dilute and condensed phases. We demonstrate the robust performance of this 1D 1H NMR experiment using biphasic samples containing condensates of FUS N-terminal domain, FUS full-length, and the multicomponent condensate formed by two intrinsically disordered regions of hnRNPC1 protein with highly overlapping resonances. Integrating the multiplexing filter into multidimensional experiments enables site-specific structural and dynamic insights from diverse protein populations, extending NMR applications in LLPS.

biophysics↗

bioRxiv2024

LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels

Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize protein, RNA and water in biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between the condensed and dispersed phases to allow the determination of not only diffusion constants in both phases but also the fractions of the species, the average radius of the condensed droplets and the exchange rate between the phases. We can also access the concentration of proteins in both phases. Observing proteins, RNAs, water, and even small molecules, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones. In addition, REDIFINE proved to be valuable beyond LLPS for the determination of binding constants in soluble protein-RNA without the need for titration.

biophysics↗

bioRxiv2022

Octopamine metabolically reprograms astrocytes to confer neuroprotection against α-synuclein

Octopamine is a well-established invertebrate neurotransmitter involved in fight-or-flight responses. In mammals, its function was replaced by norepinephrine. Nevertheless, it is present at trace amounts and can modulate the release of monoamine neurotransmitters by a yet unidentified mechanism. Here, through a multidisciplinary approach utilizing in vitro and in vivo models of -synucleinopathy, we uncovered an unprecedented role for octopamine in driving the conversion from toxic to neuroprotective astrocytes in the cerebral cortex by fostering aerobic glycolysis. Physiological levels of neuron-derived octopamine act on astrocytes via a TAAR1-Orai1-Ca2+-calcineurin-mediated signaling pathway to stimulate lactate secretion. Lactate uptake in neurons via the MCT2-calcineurin-dependent pathway increases ATP and prevents neurodegeneration. Pathological increases of octopamine caused by -synuclein halts lactate production in astrocytes and short-circuits the metabolic communication to neurons. Our work provides a novel function of octopamine as a modulator of astrocyte metabolism and subsequent neuroprotection with implications to -synucleinopathies.

neuroscience↗

bioRxiv2022

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/507991v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@183d480org.highwire.dtl.DTLVardef@1f4235aorg.highwire.dtl.DTLVardef@13d1aedorg.highwire.dtl.DTLVardef@b2c87f_HPS_FORMAT_FIGEXP M_FIG C_FIG The ongoing pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further drug development.

biophysics↗

bioRxiv2022

Integrative solution structure of a PTBP1-viral IRES complex reveals strong compaction and ordering with residual conformational flexibility

RNA-binding proteins (RBPs) are crucial regulators of gene expression and often comprise well-defined domains interspersed by flexible, intrinsically disordered regions. The structure determination of ribonucleoprotein complexes involving such RBPs is not common practice and requires integrative structural modeling approaches due to the fact that they often do not form a single stable globular state. Here, we integrate data from magnetic resonance, mass spectrometry, and small angle scattering to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1 also called hnRNP I) bound to an RNA which is part of the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). PTBP1 binding to this IRES element enhances translation of the viral RNA. The determined structural ensemble reveals that both RNA and protein experience a strong compaction upon complex formation, get ordered but still maintain a substantial conformational flexibility. The C-terminal RNA recognition motif (RRM4) of PTBP1 rigidifies the complex by binding a single-strand RNA linker and, in turn, is essential for IRES-mediated translation. PTBP1 acts as an RNA chaperone for the IRES, by ordering the RNA into a few discrete conformations that expose the RNA stems to the outer surface of the RNP complex for subsequent interactions with the translation machinery. The conformational diversity within this structural ensemble is likely common among RNP complexes and important for their functionality. The presented approach opens the possibility to characterize heterogeneous RNP structures at atomic level.

biochemistry↗