Biology subjects

Ozlu, N.

Publications and source records attributed to Ozlu, N..

12 recordsLinked to original sources

bioRxiv2026

Proximity labeling reveals cell cycle-specific NEK2 interactions and a regulatory axis controlling NUSAP1 stability

NEK2 is a cell cycle-regulated kinase best known for its role in centrosome separation, yet the phase-specific organization of its interaction network has remained unclear. Here, we combine a doxycycline-inducible TurboID system with mass spectrometry to generate a cell cycle-resolved NEK2 interactome in synchronized U2OS cells. Using generalized additive models (GAMs), we identified different enrichment trajectories of the NEK2 interacting proteins across G1/S, late S, and G2/M, linking NEK2 to chromosome and spindle regulation, RNA-ribonucleoprotein processes, vesicle/lysosome compartments, and ubiquitin-associated pathways. Targeted validations (streptavidin pull-down, co-immunoprecipitation, and immunofluorescence) confirmed the interaction and binding for selected partners. Focusing on NUSAP1, NEK2 induction led to rapid loss of NUSAP1 protein without changes in mRNA levels, and this decrease was blocked by the proteasome inhibitor MG-132. Consistently, NUSAP1 exhibited slower decay in cycloheximide chase assays and reduced ubiquitination in NEK2 knockout cells, indicating NEK2-dependent proteasomal turnover. Global proteomic analysis of NEK2-deficient cells revealed widespread remodeling of protein abundance, including increased NUSAP1 and decreased KIF2C, accompanied by coordinated changes in pathways governing mitotic progression, microtubule organization, and ubiquitin-mediated protein turnover. Together, these findings provide a dynamic map of the NEK2 interactome across the cell cycle and uncover a NEK2-NUSAP1 degradation pathway, offering a framework to study how kinase interactomes are remodeled by cell cycle progression.

cancer biology↗

bioRxiv2025

Keratin 15 promotes a progenitor cell statein basal keratinocytes of skin epidermis

The type I intermediate filament proteins keratin 14 (K14) and keratin 15 (K15) are common to all complex epithelia. K14 is highly expressed by progenitor keratinocytes, in which it provides essential mechanical integrity and gates keratinocyte entry into differentiation by sequestering YAP1, a transcriptional effector of Hippo signaling, to the cytoplasm. K15 has long been used as a marker of hair bulge stem cells, though its specific role in skin epithelia is unknown. Here we show that the lack of two biochemical determinants, a cysteine residue within the stutter motif of the central rod domain and a 14-3-3 binding site in the N-terminal head domain, renders K15 unable to effectively sequester YAP1 in the cytoplasm like K14 does. We combine insight obtained from cell culture and transgenic mouse models with computational analyses of transcriptomics data and propose a model in which a higher K15:K14 ratio promotes a progenitor state and antagonizes differentiation in keratinocytes of the epidermis. SUMMARYThe type I intermediate filament keratin 15 (K15) exhibits preferential expression in a subset of skin keratinocytes with stem-like properties. We report that K15 promotes the progenitor state by counteracting the ability of the related K14 to negatively regulate YAP1, a terminal effector of Hippo signaling.

developmental biology↗

bioRxiv2024

Keratin 16 spatially inhibits type I interferon responses in stressed skin

The stress-induced keratin 16 is broadly used as a biomarker in inflammatory skin disorders while pathogenic variants in KRT16 cause pachyonychia congenita (PC), a condition in which differentiation and homeostasis are disrupted in palmoplantar epidermis and epithelial appendages. How K16 impacts these disorders at a molecular level is poorly understood. Here we report that K16 spatially restricts type I interferon (IFN) signaling and innate immunity in palmoplantar keratoderma (PPK) lesions in PC patients, imiquimod- and phorbol ester-induced models of sterile inflammation in mouse skin, and poly(I:C)-treated human keratinocytes ex vivo. Mechanistically, K16 interacts with effectors of the RIG-I-like receptor (RLR) pathway, including 14-3-3{varepsilon}, and inhibits the 14-3-3{varepsilon}:RIG-I interaction upstream of IFN activation. Topical application of the JAK inhibitor Ruxolitinib reduces the severity of PC-PPK-like lesions in Krt16 null mice. These findings uncover a new paradigm for keratin-dependent regulation of innate immunity and suggest a new approach to PC treatment. One sentence summaryKRT16 negatively regulates type I interferon signaling and innate immune responses in the skin, offering insight into the pathophysiology of inflammatory skin diseases including pachyonychia congenita, psoriasis and others.

immunology↗

bioRxiv2023

TW68, Cryptochromes stabilizer, regulates fasting blood glucose level in ob/ob and fat-induced diabetic mice

Cryptochromes (CRYs), transcriptional repressors of the circadian clock in mammals, inhibit cAMP production when glucagon activates G-protein coupled receptors. Therefore, molecules that modulate CRYs have the potential to regulate gluconeogenesis. In this study, we discovered a new molecule called TW68 that interacts with the primary pockets of mammalian CRY1/2, leading to reduced ubiquitination levels and increased stability. In cell-based circadian rhythm assays using U2OS:Bmal1-dLuc cells, TW68 extended the period length of the circadian rhythm. Additionally, TW68 decreased the transcriptional levels of two genes, Phosphoenolpyruvate carboxykinase 1 (PCK1) and Glucose-6-phosphatase (G6PC), which play crucial roles in glucose biosynthesis during glucagon-induced gluconeogenesis in HepG2 cells. Oral administration of TW68 in mice showed good tolerance, a good pharmacokinetic profile, and remarkable bioavailability. Finally, when administered to fasting ob/ob and fat-induced diabetic animals, TW68 reduced blood glucose levels by enhancing CRY stabilization and subsequently decreasing the transcriptional levels of Pck1 and G6pc. These findings collectively demonstrate the antidiabetic efficacy of TW68 in vivo, suggesting its therapeutic potential for controlling fasting glucose levels in the treatment of type 2 diabetes mellitus.

molecular biology↗

bioRxiv2023

Aurora B Kinase Dependent Phosphorylation of Keratin 8 is required for Cytokinesis in Mammalian Cells of Epithelial Origin

Keratins assemble into mechanically resilient polymers that physically stabilize epithelial cells. When epithelial cells divide, keratin polymers must be severed to allow cell separation during cytokinesis. Phosphorylation has been implicated in this process, but how keratins are regulated during cell division is not understood. Aurora-B kinase, which is part of the chromosome passenger complex (CPC) accumulates at the cell center during cytokinesis and has been implicated in regulating intermediate filaments. We mapped six Aurora B kinase sites in Keratin 8. Phosphorylation of Keratin 8 at S34 occurred specifically at the cleavage furrow and persisted at the midzone until the completion of cytokinesis. Inhibition of Aurora B or expression of a non-phosphorylatable Keratin 8 mutant impaired keratin disassembly at the cleavage furrow. We propose that Aurora B-mediated phosphorylation promotes localized keratin filament disassembly at the cleavage furrow, allowing spatially regulated disassembly during cytokinesis. Aurora B binds to keratin filaments, and its localization to midzones was reduced in Keratin 8 knockout cells, showing that Keratin 8 facilitates Aurora B targeting during cytokinesis. This suggests a positive feedback cycle whereby Keratin 8 promotes midzone-localization of Aurora B and in turn, is locally disassembled by its kinase activity. This cycle is required for successful furrow ingression and completion of cell division in cancer cells of epithelial origin and might provide a target for solid tumor treatment.

cell biology↗

bioRxiv2021

PCDH7 Promotes Cell Migration by Regulating Myosin Activity

Cell migration requires spatiotemporally coordinated activities of multicomponent structures including the actomyosin cortex, plasma membrane, adhesion complexes and the polarity proteins. How they function together to drive this complex dynamic process remains an outstanding question. Here, we show that a member of the protocadherin family, PCDH7 displays a polarized localization in migratory cells with a dynamic enrichment at the leading and rear edges. Perturbation of PCDH7 interferes with the migration of nontransformed retinal pigment epithelial cells and the invasion of cancer cells. The overexpression of PCDH7 enhances the migration capability of cortical neurons in vivo. PCDH7 interacts with the myosin phosphatase subunits MYPT1 and PP1c{beta}. Ectopic expression of PCDH7 enhances the MYPT1 inhibitory phosphorylation levels and the phosphorylation of the myosin regulatory light chain and ERM at the polarized cortex. The chemical inhibition of phosphatase activity recovers migration phenotypes of PCDH7 knockout cells. We propose that PCDH7 regulates phosphorylation thus the activity of myosin and ERM at the polarized cortex through its interaction with myosin phosphatase. Collectively, our study suggests a new component for the spatial coordination of the plasma membrane and the cortex during cell migration.

cell biology↗

bioRxiv2021

Quantitative proteomics identifies secreted diagnostic biomarkers as well as tumor-dependent prognostic targets for clear cell Renal Cell Carcinoma

Clear cell Renal Cell Carcinoma (ccRCC) is the third most common and most malignant urological cancer, with a 5-year survival rate of 10% for patients with advanced tumors. Here, we identified 10,160 unique proteins by in-depth quantitative proteomics, of which 955 proteins were significantly regulated between tumor and normal adjacent tissues. We verified 4 putatively secreted biomarker candidates, namely PLOD2, FERMT3, SPARC and SIRP, as highly expressed proteins that are not affected by intra- and inter-tumor heterogeneity. Moreover, SPARC displayed a significant increase in urine samples of ccRCC patients, making it a promising marker for clinical screening assays. Furthermore, based on molecular expression profiles, we propose a biomarker panel for the robust classification of ccRCC tumors into two main clusters, which significantly differed in patient outcome with an almost three times higher risk of death for cluster 1 tumors compared to cluster 2 tumors. Moreover, among the most significant clustering proteins, 13 were targets of repurposed inhibitory FDA-approved drugs. Our rigorous proteomics approach identified promising diagnostic and tumor-discriminative biomarker candidates which can serve as therapeutic targets for the treatment of ccRCC.

cancer biology↗

bioRxiv2020

AF10 (MLLT10) prevents somatic cell reprogramming through regulation of H3K79 methylation

The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. Here, we employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knockout cells, re-expression wildtype AF10, but not a mutant defective in DOT1L binding, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.

developmental biology↗

bioRxiv2020

REEP4 is recruited to the inner nuclear membrane by ELYS and promotes nuclear pore complex formation

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs assemble either into the closed nuclear envelope during interphase or concomitantly with nuclear envelope reformation during anaphase. Both, interphase and post-mitotic NPC biogenesis require local deformation of membrane. Yet, the factors that control proper membrane remodeling for post-mitotic NPC assembly are unknown. Here, we report that the reticulon homology domain-protein REEP4 localizes not only to high-curvature membrane of the cytoplasmic endoplasmic reticulum (ER) but also to the inner nuclear membrane (INM). We show that REEP4 is recruited to the INM by the NPC biogenesis factor ELYS and promotes NPC assembly. REEP4 contributes mainly to anaphase NPC assembly, suggesting that REEP4 has an unexpected role in coordinating nuclear envelope reformation with post-mitotic NPC biogenesis.

cell biology↗

bioRxiv2020

Protocadherin 7 localizes to the plasma membrane during mitosis and promotes cytokinesis by a palmitoylation-dependent mechanism

Cell division requires dramatic reorganization of the cell cortex that is primarily driven by the actomyosin network. We previously reported that Protocadherin 7 (PCDH7) enriches at the cell surface during mitosis which is required for building up the full mitotic rounding pressure. Here we showed that PCDH7 gets palmitoylated and interacts with the palmitoyltransferase, ZDHHC5. Both PCDH7 and ZDHHC5 co-localize at the mitotic cell surface, and they translocate to the cleavage furrow during cytokinesis. PCDH7s localization depends on palmitoylation activity of ZDHHC5. Loss of expression of PCDH7 impairs active RhoA and phospho-myosin levels at the cleavage furrow and increases the rate of multinucleated cells. This work uncovers a palmitoylation-dependent translocation mechanism for PCDH7 and attributes a regulatory role to contributing actomyosin activity during cytokinesis.

cell biology↗

bioRxiv2020

Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation

KIF2A is a microtubule-depolymerizing kinesin motor protein with essential roles in neural progenitor division and axonal pruning during brain development. KIF2A is alternatively spliced in nervous tissue by specific RNA-binding proteins. However, how different KIF2A isoforms function during development of the cerebral cortex is not known. Here, we focus on three Kif2a isoforms expressed in mouse embryonic and postnatal cerebral cortex. We show that KIF2A is essential for dendritic pruning of primary cortical neurons in mice and that the functions of all three isoforms are sufficient for this process. Interestingly, only two of the isoforms can sustain radial migration of cortical neurons while a third isoform, lacking a key stretch of twenty amino acids, is ineffective. By proximity-labeling-based interactome mapping for individual KIF2A isoforms, we provide novel insight into how isoform specific interactions can confer changes to KIF2A protein function. Our interactome mapping identifies previously known KIF2A interactors, proteins localized to the mitotic spindle poles, and unexpectedly, also translation factors, ribonucleoproteins and proteins that are targeted to the mitochondria and ER, suggesting a novel transport function for KIF2A.

developmental biology↗

bioRxiv2019

NEUROD2 represses Reelin expression and controls dendrite orientation during cortical radial migration

Excitatory neurons of the mammalian cerebral cortex are organized into six functional layers characterized by unique patterns of connectivity, as well as distinctive physiological and morphological properties. Cortical layers appear after a highly regulated migration process in which cells move from the deeper, proliferative zone toward the superficial layers. Importantly, defects in this radial migration process have been implicated in neurodevelopmental and psychiatric diseases. Here we report that during the final stages of migration, transcription factor Neurogenic Differentiation 2 (Neurod2) contributes to terminal cellular localization within the cortical plate. In mice, in utero knockdown of Neurod2 results in reduced numbers of neurons localized to the uppermost region of the developing cortex, also termed the primitive cortical zone. Our ChIP-Seq and RNA-Seq analyses of genes regulated by NEUROD2 in the developing cortex identify a number of key target genes with known roles in Reelin signaling, a critical regulator of neuronal migration. Our focused analysis of regulation of the Reln gene, encoding the extracellular ligand REELIN, uncovers NEUROD2 binding to conserved E-box elements in multiple introns. Furthermore, we demonstrate that knockdown of NEUROD2 in primary cortical neurons results in a strong increase in Reln gene expression at the mRNA level, as well as a slight upregulation at the protein level. These data reveal a new role for NEUROD2 during the late stages of neuronal migration, and our analysis of its genomic targets offer new genes with potential roles in cortical lamination.

neuroscience↗