Biology subjects

Pavic, K.

Publications and source records attributed to Pavic, K..

6 recordsLinked to original sources

bioRxiv2025

Modulation of CIP2A dimerization and protein stability via druggable pocket

Cancerous inhibitor of PP2A (CIP2A) supports malignant growth across several cancer types. CIP2A is also a synthetic lethal therapy target for BRCA-mutant cancers. In addition, CIP2A causes Alzheime[r]s disease (AD) phenotype in mice. However, it is not dispensable for normal development and growth. Therefore, CIP2A is very lucrative therapy target in cancer and AD. So far it has been unclear whether the CIP2A protein harbors any druggable pockets amenable to targeting by small molecules. Here we discover a druggable pocket adjacent to the homodimerization domain of CIP2A. Mutagenesis demonstrates that pocket impacts CIP2A homodimerization, PP2A interaction, and full-length CIP2A protein stability in cancer cells. Further, we identify Gambogenic acid (GNA) as a tool compound that directly binds to CIP2A pocket region. GNA and its derivative Gambogic acid dissociate CIP2A homodimer in vitro and cause CIP2A destabilization in cellulo. The pocket contributes to the impact of GNA on homodimerization based on the protein fragment and mutagenesis analysis. These results identify structural vulnerability on CIP2A and may facilitate development of targeted CIP2A inhibitors with broad disease applicability.

biochemistry↗

bioRxiv2025

Proteomics- and BRET-screens identify SPRY2 as Ras effector that impacts its membrane organisation

K-Ras functions within nanoscale proteo-lipid domains of the plasma membrane, but few regulators of its membrane organisation are known. We combined TurboID-based proximity proteomics with a secondary BRET screen to identify eight novel K-Ras G-domain interactors. We focused on APLP2 and SPRY2 for further characterisation. APLP2 binds K-Ras indirectly via C-Raf, while SPRY2 exhibits properties of a novel effector. Co-immunoprecipitation and BRET assays revealed that the SPRY2 C-terminal fragment (residues 161-315) binds oncogenic RasG12V more strongly than the full-length protein. Both forms localise to the plasma membrane, but this localisation and binding to K-Ras is disrupted by inhibitors of K-Ras membrane anchorage or activity. Mutations at the predicted interface of K-Ras and SPRY2s C-terminal region affect the interaction. Both full-length SPRY2 and its C-terminal fragment promote differentiation of C2C12 muscle cells, a process requiring MAPK pathway inhibition. Finally, SPRY2 also forms homo- or hetero-oligomers with SPRY4. We propose that active K-Ras recruits SPRY2 dimers to the membrane, where they bind Ras and block effector access.

biochemistry↗

bioRxiv2024

A general Bioluminescence Resonance Energy Transfer (BRET) protocol to measure and analyze protein interactions in mammalian cells

Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here we provide a step-by-step protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe how to seed, transfect and replate cells, followed by their preparation for BRET-measurements on a microplate reader and detailed data analysis steps. For details on how to apply this protocol, please refer to Steffen et al., 2024 1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/602189v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@152e8c2org.highwire.dtl.DTLVardef@2f2aacorg.highwire.dtl.DTLVardef@9a8e9borg.highwire.dtl.DTLVardef@108833f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Identification of an H-Ras nanocluster disrupting peptide

The Ras-MAPK pathway is critical to regulate cell proliferation and differentiation. Its dysregulation is implicated in the onset and progression of numerous types of cancers. To be active, Ras proteins are membrane anchored and organized into nanoclusters, which realize high-fidelity signal transmission across the plasma membrane. Nanoclusters therefore represent potential drug targets. However, targetable protein components of signalling nanoclusters are poorly established. We previously proposed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering by stabilizing stacked dimers of H-Ras and Raf via a direct interaction of dimeric Gal1 with the Ras binding domain (RBD) in particular of B-Raf. Here, we provide further supportive evidence for this model. We establish that the B-Raf preference emerges from divergent regions of the Raf RBDs that were proposed to interact with Gal1. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B-Raf-RBD. Its 23-mer core fragment is thus sufficient to interfere with Gal1-enhanced H-Ras nanocluster, reduce MAPK-output and cell viability in HRAS-mutant cancer cell lines. Our data therefore suggest that the interface between Gal1 and the RBD of B-Raf can be targeted to disrupt Gal1-enhanced H-Ras nanoclustering. Collectively, our results support that Raf-proteins are integral components of active Ras nanoclusters.

biochemistry↗

bioRxiv2023

An improved PDE6D inhibitor combines with Sildenafil to synergistically inhibit KRAS mutant cancer cell growth

The trafficking chaperone PDE6D (or PDE) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl-binding pocket. These inhibitors suffered from low solubility and intracellular potency, preventing their clinical development. Here we developed a highly soluble PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the currently lowest off-target activity, as we demonstrate in dedicated assays. We further increased the K-Ras focus, by exploiting that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, the combination of Deltaflexin3 with the approved PKG2-activator Sildenafil synergistically inhibits cell- and microtumor growth. However, the overall cancer survival of the high PDE6D/ low PKG2 target population is higher than of the group with the opposite signature. Our results therefore suggest re-examining the interplay between PDE6D and K-Ras in cancer, while recommending the development of PDE6Di that plug, rather than stuff the hydrophobic pocket of PDE6D. SignificanceCombinations of a novel PDE6D inhibitor with Sildenafil synergistically focus the inhibition on K-Ras, however, survival data of the target population suggest an interplay of K-Ras and PDE6D that needs further exploration.

biochemistry↗

bioRxiv2020

Discovery of NOvel CIP2A VAriant (NOCIVA) and its clinical relevance in myeloid leukemias

Cancerous inhibitor of PP2A (CIP2A) is a prevalent human oncoprotein that inhibits tumor suppressor PP2A-B56a. However, CIP2A mRNA and protein variants remain uncharacterized. Here, we report discovery of a CIP2A splicing variant NOCIVA (NOvel CIp2a VAriant). NOCIVA contains CIP2A exons 1-13 fused to a continuous stretch of 349 nucleotide from CIP2A intron 13. Intriguingly, the first 39 nucleotides of the NOCIVA specific sequence are in coding frame with exon 13 of CIP2A, and codes for a 13 amino acid peptide tail unhomologous to any known human protein sequence. Therefore, NOCIVA translates to a unique human protein. NOCIVA retains the capacity to bind to B56a, but whereas CIP2A is predominantly a cytoplasmic protein, NOCIVA translocates to nucleus. Indicative of prevalent alternative splicing from CIP2A to NOCIVA in myeloid malignancies, acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) patient samples overexpress NOCIVA, but not CIP2A mRNA. In AML, high NOCIVA mRNA expression is a marker for adverse overall survival. In CML, high NOCIVA expression associates with inferior event free survival among imatinib treated patients, but not among patients treated with dasatinib or nilotinib. Collectively, we describe discovery of a novel variant of oncoprotein CIP2A, and its clinical relevance in myeloid leukemias. Key PointsO_LIDiscovery and characterization of a first mRNA variant of one of the most prevalently deregulated human oncoproteins CIP2A C_LIO_LIUnlike CIP2A, NOCIVA mRNA is overexpressed in AML and CML patient samples and associates with poor clinical response in both myeloid cancers C_LI

cancer biology↗