Biology subjects

Garvanska, D.

Publications and source records attributed to Garvanska, D..

6 recordsLinked to original sources

bioRxiv2025

A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

Serine/Threonine phosphoprotein phosphatases (PPPs, PP1-PP7) are conserved metalloenzymes and central to intracellular signaling in eukaryotes, but the details of their regulation is poorly understood. To address this, we performed genome-wide CRISPR knockout and focused base editor screens in PPP perturbed conditions to establish a high-resolution functional map of PPP regulation that pinpoints novel regulatory mechanisms. Through this, we identify the orphan reductase CYB5R4 as an evolutionarily conserved activator of PP4 and PP6, but not the closely related PP2A. Heme binding is essential for CYB5R4 function and mechanistically involves the reduction of the metal ions in the active site. Importantly, CYB5R4-mediated activation of PP4 is critical for cell viability when cells are treated with DNA damage-inducing agents known to cause oxidative stress. The discovery of a dedicated PPP reductase points to shared regulatory principles with protein tyrosine phosphatases, where specific enzymes dictate activity by regulating the active site redox state. In sum, our work provides a resource for understanding PPP function and the regulation of intracellular signaling.

cell biology↗

bioRxiv2024

A proteome-wide dependency map of protein interaction motifs

Short linear motifs (SLiMs) are the most ubiquitous protein interaction modules within unstructured regions of the human proteome, yet their contribution to cellular homeostasis remains poorly understood. To systematically assess SLiM function, we applied base editing to mutate all reported and a set of computationally predicted SLiMs defined by SLiM-like evolutionary patterns. By screening 7,293 SLiM-containing regions with 80,473 mutations in HAP1 cells, we define a SLiM dependency map identifying 450 known and 264 predicted SLiMs required for normal cell proliferation. Mutational consequences were highly reproducible in RPE1 cells, with differences attributed to cell line-specific gene essentiality. We show that most essential predicted SLiMs belong to novel classes and identify binding partners for several of these, providing mechanistic insight into a disease-associated ANKRD17 mutation. Our study provides a proteome-wide resource on SLiM essentiality and uncovers numerous uncharacterized yet essential SLiMs.

cell biology↗

bioRxiv2024

Substrate recognition principles for PP2A-B55

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modelling with comprehensive high resolution mutational scanning, we show that -helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these -helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting complex by binding to an -helical recruitment module in RBM7. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 in health and disease. One sentence summary-helices in PP2A-B55 substrates bind a conserved pocket on B55 through a common mechanism that is conserved in eukaryotes.

cell biology↗

bioRxiv2023

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

microbiology↗

bioRxiv2023

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling

Accurate chromosome segregation is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), to inhibit the anaphase-promoting complex or cyclosome (APC/C). Cdc20 is an essential mitotic regulator since it promotes mitotic exit through activating the APC/C and monitors kinetochore-microtubule attachment through activating the SAC. The proper functioning of Cdc20 requires multiple interactions with APC/C and MCC subunits. To functionally assess each of these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNAi. Here we generated Cdc20 RNAi sensitive cell lines by CRISPR/Cas9 which display a penetrant metaphase arrest phenotype by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron was found to be critical for the SAC by promoting the MCC formation and stabilizing the interaction between the MCC and APC/C. These data reveal additional regulatory components within the SAC and establish a novel method to interrogate Cdc20 function.

cell biology↗

bioRxiv2023

Global substrate identification and high throughput in vitro dephosphorylation reactions uncover PP1 and PP2A-B55 specificity principles

Phosphoprotein phosphatases (PPPs) dephosphorylate Serine (Ser)/Threonine (Thr) residues to regulate major signaling pathways and cellular transitions. Despite the central role of PPPs the substrates in most cellular processes and the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A- B55, which reveals common and unique preferences for the two phosphatases. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with quantitative phosphoproteomics to identify more than 2000 regulated phosphorylation sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with the MRBLE:Dephos results. We use these insights to specifically alter INCENP dephosphorylation kinetics at mitotic exit, resulting in defective cytokinesis thus underscoring the biological relevance of our determined specificity principles. Finally, we provide a comprehensive characterization of PP1 binding motifs and demostrate how binding of phosphatases to substrates shapes dephosphorylation specificity. Collectively, we develop novel approaches to advance our ability to investigate protein phosphatases and use these to provide a framework for understanding mitotic exit regulation by dephosphorylation.

cell biology↗