Biology subjects

Zivkovic, D.

Publications and source records attributed to Zivkovic, D..

11 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into the role of Cysteine-Rich Receptor-Like Kinase 18 (CRK18) in Arabidopsis

Plants perceive and integrate diverse environmental signals through receptor kinase (RK) networks at the plasma membrane. Within this, Cysteine-Rich Receptor-Like Kinases (CRKs) constitute a large but not well-understood subfamily characterised by extracellular domains (ECDs) enriched in cysteine residues. CRKs have been implicated in plant responses to biotic and abiotic stress, as well as in developmental processes. Additionally, several CRKs have been proposed to act as redox sensors. Here, we investigate the homodimerization mechanism of Arabidopsis CRK18 and its regulation by redox conditions. By modulating pH and redox state, we assessed the stability and binding dynamics of the CRK18 ECD and its cysteine mutants. We also tested the plasma membrane localisation of all the cysteine mutants involved in the predicted disulfide bonds, and only CRK18C227A, C228A-ECD resembled the plasma membrane localization of wild-type CRK18. We combine co-immunoprecipitation, Forster resonance energy transfer-fluorescence lifetime imaging microscopy and microscale thermophoresis to quantify CRK18 self-association in planta and in vitro. Furthermore, we place CRK18 dimerization in a broader signalling context by identifying CRK18 interaction partners and CRK18-dependent signalling outputs. Using Arabidopsis CRK18 overexpression lines, we perform (phospho)proteomic and immunoprecipitation-mass spectrometry (IP-MS) analyses to map CRK18-centred signalling networks associated with stress-related responses. The constitutive activation of the CRK18 kinase domain and its interaction with many putative (cell wall) glycan-sensing RKs, including PERK15, suggest a regulatory role for CRK18. The absence of significant changes in the proteome and phosphoproteome of CRK18 overexpression lines in the absence of any trigger, and its restricted mobility after elicitation, suggest that CRK18 requires a stimulus for activation and possibly induces membrane microdomain reorganisation. This is in line with the infection assay with the nematode Heterodera schachtii, which causes modification and targeted damage to plant cell walls during infection, revealing that CRK18 acts as negative regulator of this process.

biochemistry↗

bioRxiv2026

GAPMs form a heterotrimeric complex bridging the gliding machinery and the cytoskeleton across Plasmodium species

Apicomplexan parasites, such as malaria-causing Plasmodium spp., use a specialised actomyosin motor system known as the glideosome to drive movement through host tissue and invade host cells. This system is anchored to the inner membrane complex (IMC), a series of flattened vesicles located beneath the plasma membrane, and thought to be linked to the underlying cytoskeleton by the GAPM protein family. However, it is not known how these GAPM proteins are localised across the Plasmodium life cycle, and whether different family members function alone or together. Here, we show that in two Plasmodium species GAPM2 is an IMC component whose recruitment and organisation are tightly coordinated with nuclear and cytoskeletal dynamics during parasite replication and differentiation. We find that the GAPM2 interactome remodels between asexual and sexual stages using mass spectrometry. To understand the molecular relationship between three GAPM paralogues, we solved a cryo-electron microscopy structure of the GAPM complex. This revealed an obligate heterotrimeric architecture that forms an asymmetric platform, likely to serve as a docking interface for other components of the glideosome. Finally integrating our GAPM heterotrimer structure with mass spectrometry data allowed us to propose a unified structural model of the glideosome that is conserved across apicomplexan parasites.

microbiology↗

bioRxiv2025

KDM7-mediated oxygen sensing reprograms chromatin to enhance hypoxia tolerance in the root

Roots frequently encounter low oxygen (hypoxia) from soil compaction or water saturation and must adapt to this stress. We investigated how root tip cells sense hypoxia and adjust the meristem epigenome to activate genes that promote tolerance and growth under oxygen limitation. In Arabidopsis root tips, hypoxia tolerance was linked to increased trimethylation of histone H3 at lysine 4 (H3K4me3). We also found that group 7 demethylases (KDM7s) are directly inhibited by hypoxia, and that genetic inactivation of KDM7s, like hypoxia, induces expression of genes essential for meristem survival under oxygen deprivation. We propose that KDM7s function as root-specific oxygen sensors that prime and support hypoxia tolerance.

plant biology↗

bioRxiv2025

Revealing the unexpected interplay between the Proteasome Activator PA200 and the immunoproteasome

The proteasome activator PA200 binds to the catalytic core of the proteasome, the 20S, and activates its proteolytic activities. The cellular function of PA200 is poorly understood and appears to be cell type and differentiation specific. Recent evidence suggests that PA200 not only binds to the standard 20S (s20S) proteasome but also to the specialized immunoproteasome (i20S) which plays a key role in anti-viral and anti-tumor immunity. We here investigated the interaction of PA200 and the immunoproteasome in detail. We show the very first cryo-EM structures of the singly- and doubly-capped i20S-PA200 complexes that revealed no major difference regarding the first binding event of PA200 to the i20S vs. the s20S. However, first PA200 binding triggered a subtle and long range allosteric bending of the i20S barrel which was not seen in the s20S-PA200 complexes. This resulted in major structural rearrangements in the opposite unbound ring - the displacement of atoms up to 5.4 [A] and the increase in its outer diameter - thereby increasing the occupancy of the second PA200 binding site. Mass photometry confirmed higher occupancy of PA200 to the i20S versus the s20S. Binding of PA200 to the i20S enhanced proteasomal activation compared to the s20S. Co-expression of PA200 and the i20S in cells and tissues, however, is restricted but their interaction is favored upon co-expression. The expression of PA200 and the catalytic subunits of the i20S is differentially regulated depending on the cellular context. Our data also suggest that PA200 has the potential to regulate i20S gene expression whereas the i20S has no effects on PA200 expression. Overall, this work sheds new light on the interaction of PA200 with the i20S from a structural, mechanistic and cellular point of view. Importantly, we identify PA200 as a key regulator of the i20S whenever PA200 and the catalytic subunits of the i20S are co-expressed in the same cell.

biochemistry↗

bioRxiv2024

The proteasome activator PA200 regulates tumor cell invasion

Proteasome activator 200 (PA200) is upregulated in non-small cell lung cancer (NSCLC) and linked to poor prognosis. We previously demonstrated that the overexpression of PA200 in NSCLC is associated with immune evasion and reduced responsiveness to immune checkpoint inhibitors. The cell autonomous function of PA200 in cancer growth, however, is not solved. We here demonstrate that deletion of PA200 in two distinct lung cancer cell lines induced cell-specific alterations in proteasome composition and activities with a minor direct impact on overall proteasome activity. Deficiency of PA200 in lung cancer cells did not consistently alter tumor cell growth in vitro and in vivo. However, we observed concerted inhibition of tumor cell migration and invasion with conserved downregulation of the integrin ITGB3 and transcriptional dysregulation of multiple cell adhesion and ECM regulators. Our transcriptome profiling revealed a striking disparity in the transcriptional response to PA200 deletion in the two lung cancer cell lines. Together with our PA200 interactome analysis that uncovered an unexpected cell-dependent profile of PA200-interacting proteins, our data indicate that the function of PA200 is cell-specific and depends on the cellular context. In conclusion, we here demonstrate that PA200 cell-autonomously regulates the invasive capacities of tumor cells thereby potentially promoting lung cancer spread and metastasis formation. This mechanism might add to PA200-related immune evasion and may contribute to the observed poor prognosis of PA200-overexpressing lung cancer patients.

cancer biology↗

bioRxiv2024

MAASTY: A (dis)ordered copolymer for structural determination of human membrane proteins in native nanodiscs

Amphiphilic copolymers capable of extracting membrane proteins directly from cellular membranes into "native nanodiscs" offer a simplified approach for preparing membrane proteins in lipid nanodiscs compared to approaches that rely on detergent. Amphiphilicity, length, and composition influence the performance of copolymers, in addition to the protein itself and the purification conditions used. Here, we report a copolymer composed of methacrylic acid and styrene, which we term MAASTY, leveraging the inherent monomer reactivity ratios to create an anionic copolymer with a statistical distribution of monomers. We show that MAASTY can be used for high-resolution structural determination of a human membrane protein with single particle cryo-electron microscopy, preserving endogenous lipids including cholesterol and exhibiting an enrichment of phosphatidylinositol. Moreover, MAASTY copolymers effectively solubilize a broad range of lipid species and a wide range of different, eukaryotic membrane proteins from mammalian cells. We find that MAASTY copolymers are promising as effective solubilizers of membrane proteins and offer a chemical platform for structural and functional characterization of membrane proteins in native nanodiscs.

biochemistry↗

bioRxiv2021

Interactome analyses reveal profound proteasome structural and functional rearrangements throughout mammalian spermatogenesis

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific 4s subunit replaces the 4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82-92%) during differentiation, presumably due to the shift from 4 to 4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S, and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between 4 and 4s. We propose that these differences induced by 4s incorporation result in significant changes in the way the s20S interacts with its partners, and dictate its role in germ cell differentiation.

biochemistry↗

bioRxiv2020

Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependant inter-ring crosstalks

Here, we used for the first time Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28{beta} or PA28{gamma} activators. Their solvent accessibility was analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirmed the existence of allosteric differences between the std20S and i20S at the surface of the -ring triggered from inside the catalytic {beta}-ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modified solvent accessibility due to conformational changes of the {beta}-rings. This work is not only a proof-of-concept that HDX-MS can be used to get structural insights on large multi-protein complexes in solution, it also demonstrates that the binding of the std20S or i20S subtype to any of its PA28 activator triggers allosteric changes that are specific to this 20S/PA28 pair.

biophysics↗

bioRxiv2019

The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, non-toxic concentrations of interleukin 1 b

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic {beta} cells, as this might facilitate autoantigen presentation by {beta} cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in {beta} cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes.\n\nWe show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the {beta}5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by non-toxic concentrations of IL-1{beta} stimulating proinsulin biosynthesis. These findings suggest that the {beta} cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

molecular biology↗

bioRxiv2019

Neutral genomic signatures of host-parasite coevolution.

Coevolution is a selective process of reciprocal adaptation between antagonistic or mutualistic symbionts and their host. Classic population genetics theory predicts the signatures of selection at the interacting loci but not the neutral genome-wide polymorphism patterns. We here build a coevolutionary model with cyclic changes in the host and parasite population sizes. Using an analytical framework, we investigate if and when these population size changes can be observed in the neutral site frequency spectrum of the host and parasite full genome data. We show that polymorphism data sampled over time can capture the changes in the population size of the parasite but not of the host because genetic drift and mutations occur on different time scales in the coevolving species. This is due to the small parasite population size at the onset of the coevolutionary history subsequently undergoing a series of strong bottlenecks. We also show that tracking coevolutionary cycles is more likely for a small amount of parasite per host and for multiple parasite generations per host generation. Our results demonstrate that time sampling of host and parasite full genome data are crucial to infer the co-demographic history of interacting species.

evolutionary biology↗

bioRxiv2017

Effects of population- and seed bank noise on neutral evolution and efficacy of natural selection

Population genetics models typically consider a fixed population size and a unique selection coefficient. However, population dynamics inherently generate noise in numbers of individuals and selection acts on various components of the individuals fitness. In plant species with seed banks, the size of both the above- and below-ground compartments present noise depending on seed production and the state of the seed bank. We investigate if this noise has consequences on 1) the rate of genetic drift, and 2) the efficacy of selection. We consider four variants of two-allele Moran-type models defined by combinations of presence and absence of noise in above-ground and seed bank compartments. Time scale analysis and dimension reduction methods allow us to reduce the corresponding Fokker-Planck equation to a one-dimensional diffusive Moran model. We first show that if the above-ground noise classically affects the rate of genetic drift, below-ground noise reduces the diversity storage effect of the seed bank. Second, we consider that selection can act on four different components of the plant fitness: plant or seed death rate, seed production or seed germination. Our striking result is that the efficacy of selection for seed death rate or germination rate is reduced by seed bank noise, whereas selection occurring on plant death rate or seed production is not affected. We derive the expected site-frequency spectrum reflecting this heterogeneity in selection efficacy between genes underpinning different plant fitness components. Our results highlight the importance to consider the effect of ecological noise to predict the impact of seed banks on neutral and selective evolution.

evolutionary biology↗