Biology subjects

Wysocki, V.

Publications and source records attributed to Wysocki, V..

7 recordsLinked to original sources

bioRxiv2025

Mechanism of SARS-CoV-2 Nucleocapsid Protein Phosphorylation-induced Functional Switch

The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed WT Np formed discrete complexes on viral 5' UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1-4. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs. WT Np also compacted and irreversibly bound single-stranded DNA; this activity was significantly reduced by phosphorylation. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants.

biochemistry↗

bioRxiv2024

Novel multiparametric bulk and single EV pipeline for adipose cell-specific biomarker discovery in paired human biospecimens

Obesity remains a growing and global public health burden across a broad spectrum of metabolic, systemic, and neurodegenerative diseases. Previously considered merely a fat storage depot, adipose tissue is now recognized as an active endocrine organ crucial for metabolic and systemic regulation of local and distant organs. A burgeoning line of investigation centers on adipose-derived extracellular vesicles (ADEVs) and their pivotal role in obesity-associated pathobiology. However, robust methodologies are lacking for specifically isolating and characterizing human ADEVs. To bridge this gap, we have developed a robust multiparametric framework incorporating bulk and single EV characterization, proteomics, and mRNA phenotyping. EVs from matched human visceral adipose tissue, mature adipocyte-conditioned media, and plasma collected from the same individual bariatric surgical patients were analyzed and subjected to bottom-up proteomics analysis. This framework integrates bulk EV proteomics for cell-specific marker identification and subsequent single EV interrogation with single-particle interferometric reflectance imaging (SP-IRIS) and total internal reflection fluorescence (TIRF) microscopy. Our proteomics analysis revealed 76 unique proteins from adipose tissue-derived EVs (ATEVs), 512 unique proteins from adipocyte EVs (aEVs), and 1003 shared proteins. Prominent pathways enriched in ATEVs included lipid metabolism, extracellular matrix organization, and immune modulation, while aEVs exhibited enhanced roles in chromatin remodeling, oxidative stress responses, and metabolic regulation. Notably, adipose tissue-specific proteins such as adiponectin and perilipin were highly enriched in ADEVs and confirmed in circulating plasma EVs. Colocalization of key EV and adipocyte markers, including CD63 and PPARG, were validated in circulating plasma EVs. In summary, our study paves the way toward a tissue and cell-specific, multiparametric framework for an adiposity EV signature in obesity-driven diseases.

molecular biology↗

bioRxiv2023

Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring a three-helix bundle, or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form has been shown to bind and inhibit TRAP. We demonstrate the utility of native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) for monitoring the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we report the use of solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP inhibition.

biochemistry↗

bioRxiv2022

Binding of ATP to GroEL: a Case for Variable-Temperature Native Mass Spectrometry Thermodynamic Studies

Understanding how large, oligomeric protein complexes respond to the binding of small ligands and other proteins is essential for describing the molecular basis of life. This in turn requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures, including effects of small molecules and solvent. However, the size of many protein oligomers, the myriad intermediate ligation states they can populate, and their often complex allosteric regulation typically restrict analysis by traditional methods to low resolution, ensemble averages. Here, we employ variabletemperature electrospray ionization native mass spectrometry to determine the thermodynamics for stepwise binding of up to ATP molecules to the 801 kDa GroEL complex, a tetradecamer chaperonin complex. Binding thermodynamics reveal strong enthalpy-entropy compensation (EEC) and high degrees of cooperativity are observed for formation of GroEL-ATP7 and GroEL-ATP14. These are evidenced by entropically favored ATP binding to the cis ring (formation of GroEL-ATP1-7), with variations in EEC for subsequent binding of ATP to the trans ring (GroEL-ATP8-14), as expected for negative inter-ring cooperativity. Entropy driven ATP binding to the GroEL tetradecamer is consistent with ligand induced conformational changes of the GroEL tetradecamer, though the magnitude of the entropy change suggests that reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity may also play a key role. By determining the thermodynamic signatures for individual ligand binding reactions to the large, nearly MDa GroEL complex, we expand our fundamental view of chaperonin functional chemistry. Moreover, this work and related studies of protein-ligand interactions illustrate unparalleled capabilities of vT-ESI-nMS for thermodynamics studies of protein interactions with ligands, and other molecules, such as proteins and drugs.

biophysics↗

bioRxiv2022

Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry

RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfqs integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry (nMS) coupled with surface-induced dissociation (SID) and molecular dynamics (MD) simulations to disentangle the arrangement of the CTDs and their impact on the stability of E. coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding. Significance StatementHfq is a protein hexamer necessary for gene regulation by non-coding RNA in bacteria, during infection or under stress. In the cell, Hfq must distinguish its RNA partners from many similar nucleic acids. Mass spectrometry dissociation patterns, together with molecular dynamics simulations, showed that flexible extensions of each Hfq subunit form a dense network that interconnects the entire hexamer. This network is disrupted by RNA binding, but the lost interactions are compensated by RNAs that contact multiple Hfq subunits. By measuring interactions that are too irregular to be counted by other methods, mass spectrometry shows how flexible protein extensions help chaperones like Hfq recognize their RNA partners in the messy interior of the cell.

biophysics↗

bioRxiv2021

Unloxing the assembly and activation mechanism of Cre recombinase using Cryo-EM

Mechanistic understanding of the structural basis for DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes (intasomes). These structural and biochemical studies have suggested that conformational changes and DNA bending in presynaptic complexes underlie site-selection and activation mechanisms of Cre recombinase. Here we used protein engineering and various DNA substrates to isolate the Cre-loxP (54 kDa), Cre2-loxP (110 kDa), and Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9 [A], 4.5 [A], and 3.2 [A], respectively. Progressive DNA bending along the assembly pathway enables formation of increasingly intimate protein-protein interfaces. Insufficient stabilization of important protein motifs observed during the assembly process provides a compelling explanation for the observed half-the-sites activity, and preferential bottom strand cleavage of loxP sequences. We found that selection of loxP sites is largely dependent on Cres ability to bend and stabilize the spacer region between two recombinase binding elements. Application of 3D variability analysis to the tetramer data reveals a propensity for motion along the pathway between protomer activation and Holliday junction isomerization. These findings help us to better understand loxP site specificity, controlled activation of alternating protomers, the basis for the observed bias of strand cleavage order, and the importance of conformational sampling, especially with regards to site-selection and activity among Cre variants. Furthermore, our findings provide invaluable information for the rational development of designer, site-specific recombinases for use as gene editing technologies. HighlightsO_LICryo-EM structures of Cre-loxP assembly intermediates (monomer, dimer, and tetramer) provide insights into mechanisms of site recognition, half-the-sites activity, strand cleavage order, and concerted strand cleavage C_LIO_LISelectivity of loxP sites arises from few base-specific contacts made by each protomer and is mainly driven by formation of phosphate contacts and DNA deformations that are maximal in the fully assembled "active" tetramer C_LIO_LICis and trans interactions of the {beta}2-3 loop (i) define which sites are "active" and (ii) ensure half-the-sites activity C_LIO_LIProtein flexibility plays a central role in enabling DNA sequence scanning, recruitment of a second protein to form a dimer, synapsis, control of activity, as well as subsequent recombination steps C_LIO_LIConformational sampling within the tetrameric complex was uncovered using 3D variability analysis and revealed the importance of protein-protein interfaces for site- selection and activation of Cre-loxP complexes C_LI

biochemistry↗

bioRxiv2018

The exon junction complex undergoes a compositional switch that alters mRNP structure and nonsense-mediated mRNA decay activity

The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that EJC originates as an SR-rich mega-dalton sized RNP that contains RNPS1 but lacks CASC3. After mRNP export to the cytoplasm and before translation, the EJC undergoes a remarkable compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general whereas CASC3 is needed for NMD of only select mRNAs. The promotion of switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.

molecular biology↗