Biology subjects

Bozkurt, E.

Publications and source records attributed to Bozkurt, E..

8 recordsLinked to original sources

bioRxiv2026

Assembly and activation of the AAA+ motor of the multi-subunit cobaltochelatase

Cobalamin is a complex yet essential cobalt-containing cofactor synthesized exclusively by prokaryotes and archaea. Aerobic cobalamin biosynthesis requires the CobNST multi-subunit class I cobaltochelatase for cobalt incorporation. Cobalt chelation is dependent on the energy transfer from the CobST AAA+/VWA motor unit to the substrate-binding catalytic unit (CobN). However, the mechanism for assembly and function of CobNST cobaltochelatase remains poorly understood. Here, we used cryo-EM to solve the structures of the AAA+ motor CobS, in the absence and presence of ATP and its VWA-partner protein CobT. Our data reveal that the AAA+ CobS assembles into various conformations, including hexameric planar rings and different spiral left-handed assemblies: hexamers, double rings and filaments. In the absence of nucleotide, the CobT N-terminal domain interacts with CobS left-handed oligomers, remaining in a half-assembled, nonfunctional motor unit. In the CobST complex, ATP induces conformational rearrangement of CobS from left-handed helical oligomers to a right-handed closed hexamer. The simultaneous repositioning of the CobT VWA domain to the AAA+ central pore shifts the motor unit to a functional state, ready-to-drive CobN-catalyzed chelation reaction. These findings define the mechanism for assembly and activation of the class I cobaltochelatase CobST motor.

biochemistry↗

bioRxiv2026

Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis

Entosis is a cell-in-cell (CIC) invasion process in which one living cell actively invades into another, with important implications for tumor evolution, cell competition, and responses to cellular stress. Both the initiation and progression of entosis require extensive actin cytoskeletal remodeling, as one cell must physically accommodate the complete internalization of another viable cell, which can subsequently migrate, divide, undergo degradation, or escape. Nearly two decades after its discovery, mechanistic insight into how cytoskeleton organization is regulated during entosis remains poorly understood. The most consistent observation is that inhibition of Rho-ROCK signaling blocks entosis, indicating a central role for actomyosin contractility. Consistently, increased phosphorylation of myosin light chain and cortical actomyosin accumulation have been reported in invading cells, yet the underlying mechanisms remain largely unknown. Here, we systematically interrogated ROCK activation as a driver of the entotic internalization program. Constitutively active mutant ROCK1 or pharmacological ROCK1 activation using narciclasine rapidly triggered canonical entosis pathway through MLC2 phosphorylation. Mass spectrometry identified Plastin-3 (PLS3, T-plastin), a crucial calcium-sensitive actin-bundling protein, which was recruited to the plasma membrane to trigger entosis upon ROCK1 activation. Our study uncovers a key cytoskeletal remodeling step which may support cortical tension required for CIC invasion during entosis.

cancer biology↗

bioRxiv2025

Multiplex analysis of colorectal cancer tissue describes the composition, cell biology and spatial effects of cell-in-cell events and identifies a T cell-dependent prognostic signature

Cell competition is an emerging mechanism in which mammalian tissues maintain homeostasis by eliminating less fit (loser) cells through direct interactions with fitter (winner) neighbouring cells. In cancer, these competitive interactions may drive tumour evolution; however, spatial organisation and clinical relevance of these events remain poorly understood. One mechanism by which winner cells eliminate loser cells is engulfment, resulting in cell-in-cell (CIC) formation. Although CICs have been observed in many tumour types for over a century, their cellular composition, spatial context, interactions with the tumour microenvironment, and biological significance in human cancers remain unclear. Here, we systematically characterised the cellular identity and functional states of CICs in situ, examined their spatial interactions within the tumour microenvironment, and assessed their clinical relevance using spatially resolved single-cell data from a large cohort of colorectal cancer patients. We demonstrate that CICs occur predominantly between cancer cells but also involve cancer stem cell (CSC)-like populations and cytotoxic T cells. Engulfed (inner) cancer and CSC-like cells display molecular features consistent with a loser-cell phenotype, including increased apoptosis and reduced proliferation, whereas outer cancer cells exhibit winner-cell features such as upregulated glycolysis. Live-cell time-lapse experiments demonstrate that glucose accumulates in inner cells during lysosomal degradation following cell engulfment. Spatial analysis further revealed distinct CIC neighbourhoods, which we defined based on proximity to engulfment events. Cells within these regions, particularly CSC-like cells and cytotoxic T cells, exhibit increased metabolic stress, suggesting local competition for nutrients. Importantly, the presence of cytotoxic T cells within CIC neighbourhoods and spatial co-occurrence patterns between cancer cells and CSC-like populations are associated with improved patient outcomes. Together, our findings demonstrate that cell engulfment defines spatially organised competitive niches and may reflect cell competition within complex tumour microenvironments.

cancer biology↗

bioRxiv2024

Androgen receptor interactions provide insight into steroid mediated metabolic shifts in endocrine resistant breast cancer.

PurposeAromatase Inhibitors (AI) are standard therapy for hormone receptor positive breast cancers in post-menopausal patients. Disease recurrence is common and previous studies suggest that the altered steroid environment may be a driver of resistance. Using label-free mass-spectrometry we explored the unique androgen receptor (AR) interactome that supervenes in AI resistant breast cancer and the associated hyperandrogenic environment. Experimental DesignAR expression was evaluated in a primary breast cancer tissue-microarray (n=875) with nuclear and cytoplasmic localization quantified. Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) analysis was utilized to identify proteins interacting with the AR in models of AI-resistance. Validation was carried out by co-immunoprecipitation and co-localisation studies. Live-cell imaging, Seahorse MitoStress Assays and flow cytometry were used to quantify changes in mitochondria and cell metabolism arising in models of AI-resistance. ResultsUtilising digital pathology we detected that abundant cytoplasmic AR protein was associated with poor survival only in the post-menopausal cohort, and most significantly, in the therapy-refractory Luminal B subtype (p=0.0085). Models of AI-resistance and androgen excess highlight diffuse AR localisation throughout the cytoplasm and nucleus accompanied by increased mitochondrial mass and membrane potential, and increased oxidative phosphorylation and glycolysis. Exploration of the AR protein interactome identified G3BP1, SLIRP, and IGFBP5 as AR protein partners which are associated with stress, adaptive metabolic response and estrogen receptor repression. ConclusionsThe findings of this study highlight the prognostic potential of cytoplasmic AR immunoreactivity in specific breast cancer subtypes and uncover novel extra-nuclear AR protein interactions that may mediate metabolic adaptations during the development of endocrine-resistance.

cancer biology↗

bioRxiv2024

Spatial Effects of Infiltrating T cells on Neighbouring Cancer Cells and Prognosis in Stage III CRC patients

Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks, as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil-based chemotherapy. Images underwent segmentation for tumour, stroma and immune cells, and cancer cell state protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell - T cell interactions at single-cell level. In our discovery cohort (MSK), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (HV) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (Discovery cohort: p = 0.07, Validation cohort: p = 0.19). We next utilized our region-based nearest neighbourhood approach to determine the spatial relationships between cytotoxic T cells, helper T cells and cancer cell clusters. In the both cohorts, we found that lower distance between cytotoxic T cells, T helper cells and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (Discovery cohort: p = 0.01, Validation cohort: p = 0.003).

cancer biology↗

bioRxiv2021

Protein Engineering with A Glycosylation Circuit Enables Improved Enzyme Characteristics

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins, making it an interesting for many biotechnology applications. The discovery of bacterial glycosylation mechanisms, opened a new perspective and transfer of C.jejuni N-linked glycosylation into laboratory work-horse E. coli increased research pace in the field exponentially. It has been previously showed that utilizing N-Linked Glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. In this study, we utilized N-linked Glycosylation to glycosylate alkaline phosphatase (ALP) enzyme in E. coli and investigate the effects of glycosylation on an enzyme. Considering the glycosylation mechanism is highly dependent on the acceptor protein, ALP constructs carrying glycosylation tag at different locations of the gene has been created and glycosylation rates have been calculated. The most glycosylated construct has been selected for comparison with the native enzyme. We investigated the performance of glycosylated ALP in terms of its thermostability, proteolytic stability, tolerance to suboptimal pH and under denaturing conditions. Studies showed that glycosylated ALP performed remarkably better at optimal and harsh conditions Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.

synthetic biology↗

bioRxiv2020

TRAIL signalling promotes entosis in colorectal cancer

Entosis is a form of non-phagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumours show evidence of entosis, however factors controlling entosis remain to be elucidated. Here we find that the death receptor ligand TRAIL is a potent activator of entosis in colon cancer cells. CLEM/3D confocal microscopy analysis revealed ultrastructural features of entosis and subsequent entotic cell death of inner cells upon TRAIL treatment. Induction of entosis and apoptosis by TRAIL were mutually exclusive events but both required the presence of caspase-8. Bax/Bak double knock-out or caspase inhibition altered the fate of inner cells from entotic cell death to survival and escape. Analysis of colorectal cancer tumours showed a significant association between expression levels of TRAIL and CICs. Notably, the presence of CICs in the invasive front regions of colorectal tumours was significantly correlated with adverse patient prognosis.

cancer biology↗

bioRxiv2020

Development of EphA2 siRNA-loaded lipid nanoparticles and combination with a small-molecule histone demethylase inhibitor in prostate cancer cells and tumor spheroids

siRNAs hold a great potential for cancer therapy, however, poor stability in body fluids and low cellular uptake limit their use in the clinic. To enhance the bioavailability of siRNAs in tumors, novel, safe, and effective carriers are needed. Here, we developed cationic solid lipid nanoparticles (cSLNs) to carry siRNAs targeting EphA2 receptor tyrosine kinase (siEphA2), which is overexpressed in many solid tumors including prostate cancer (PCa). Using DDAB cationic lipid instead of DOTMA reduced nanoparticle size and enhanced both cellular uptake and gene silencing in PCa cells. After verifying the efficacy of siEphA2-loaded nanoparticles, we further evaluated a potential combination with a histone lysine demethylase inhibitor, JIB-04. Silencing EphA2 by siEphA2-loaded DDAB-cSLN did not affect the viability (2D and 3D), migration, and clonogenicity of PC-3 cells alone. However, upon co-administration, there was a decrease in the aforementioned cellular responses due to JIB-04. Furthermore, JIB-04 decreased EphA2 expression, and thus, silencing efficiency of siEphA2-loaded nanoparticles was further increased with co-treatment. In conclusion, we have successfully developed a novel siRNA-loaded lipid nanoparticle for targeting EphA2. Moreover, detailed preliminary results of the effects of JIB-04, alone and in combination with siEphA2, on PCa cells and tumor spheroids were presented for the first time. Our delivery system provides high transfection efficiency and shows a great promise for targeting other genes and cancer types in further in vitro and in vivo studies.

pharmacology and toxicology↗