Biology subjects

Mansfeld, J.

Publications and source records attributed to Mansfeld, J..

10 recordsLinked to original sources

bioRxiv2024

Chromosome end protection by RAP1-mediated inhibition of DNA-PK

During classical non-homologous end joining (cNHEJ), DNA-dependent protein kinase (DNA-PK) encapsulates free DNA ends, forming a recruitment platform for downstream end-joining factors including Ligase 4 (LIG4)1. DNA-PK can also bind telomeres and regulate their resection2-4, but does not initiate cNHEJ at this position. How the end joining process is regulated in this context-specific manner is currently unclear. Here we show that the shelterin components TRF2 and RAP1 form a complex with DNA-PK that directly represses its end joining function at telomeres. Biochemical experiments and cryo-electron microscopy reveal that when bound to TRF2, RAP1 establishes a network of interactions with KU and DNA that prevents DNA-PK from recruiting LIG4. In mouse and human cells, RAP1 is redundant with the Apollo nuclease in repressing cNHEJ at chromosome ends, demonstrating that the inhibition of DNA-PK prevents telomere fusions in parallel with overhang-dependent mechanisms. Our experiments show that the end joining function of DNA-PK is directly and specifically repressed at telomeres, establishing a molecular mechanism for how individual linear chromosomes are maintained in mammalian cells.

biochemistry↗

bioRxiv2024

The mitotic CIP2A-TOPBP1 axis facilitates mitotic pathway choice between MiDAS and MMEJ

Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is inactivated in mitosis. Mitotic cells instead rely on the mechanisms of microhomology mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). However, how these pathways are regulated in mitosis remains unknown. Here we reveal the CIP2A-TOPBP1 complex facilitates recruitment of SMX complex components to mitotic chromatin marked by CIP2A, through a CDK1-dependent interaction between TOPBP1 BRCT 1/2 and SLX4 phospho-threonine1260, that drives MiDAS. Furthermore, CIP2A promotes the recruitment of Pol{theta} to facilitate mitotic MMEJ. This defines a mechanistic framework for mitotic DSB repair, where simultaneous disruption of MiDAS and MMEJ pathways underpins the synthetic lethality observed in BRCA1/2-deficient cells following CIP2A depletion. These findings provide critical insights into mitotic DNA repair and highlights therapeutic opportunities in HR deficient tumours.

molecular biology↗

bioRxiv2024

Cell cycle-dependent S-sulfenyl proteomics uncover a redox switch in p21-CDK feedback governing the proliferation-senescence decision

Reactive oxygen species (ROS) influence cell proliferation and fate decisions by oxidizing cysteine residues (S-sulfenylation) of proteins, but specific targets and underlying regulatory mechanisms remain poorly defined. Here, we employ redox proteomics to identify cell cycle-coordinated S-sulfenylation events and investigate their functional role in proliferation control. Although ROS levels rise during cell cycle progression, overall oxidation of the proteome remains constant with dynamic S-sulfenylation restricted to a subset of cysteines. Among these, we identify a critical redox-sensitive cysteine residue (C41) in the cyclin-dependent kinase (CDK) inhibitor p21. C41 oxidation regulates the interaction of p21 with CDK2 and CDK4, controlling a double-negative feedback loop that determines p21 stability. When C41 remains reduced, p21s half-life increases in G2 phase resulting in more p21 inheritance to daughter cells suppressing proliferation and promoting senescence after irradiation. Notably, we identify dynamic S-sulfenylation on further cell cycle regulators implying coordination of cell cycle and redox control.

cell biology↗

bioRxiv2024

High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells

The cell cycle governs a precise series of molecular events, regulated by coordinated changes in protein and phosphorylation abundance, that culminates in the generation of two daughter cells. Here, we present a proteomic and phosphoproteomic analysis of the human cell cycle in hTERT-RPE-1 cells using deep quantitative mass spectrometry by isobaric labelling. Through analysing non-transformed cells, and improving the temporal resolution and coverage of key cell cycle regulators, we present a dataset of cell cycle-dependent protein and phosphorylation site oscillation that offers a foundational reference for investigating cell cycle regulation. These data reveal uncharacterised regulatory intricacies including proteins and phosphorylation sites exhibiting previously unreported cell cycle-dependent oscillation, and novel proteins targeted for degradation during mitotic exit. Integrated with complementary resources, our data link cycle-dependent abundance dynamics to functional changes and are accessible through the Cell Cycle database (CCdb), an interactive web-based resource for the cell cycle community. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/599917v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@bb2a69org.highwire.dtl.DTLVardef@1dd55ecorg.highwire.dtl.DTLVardef@34073eorg.highwire.dtl.DTLVardef@1c7a8c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

A ROS-dependent mechanism to drive progression through S phase

Long considered as cytotoxic reagents, reactive oxygen species (ROS) at the right concentration promote cell proliferation in cell culture, stem cells and model organisms. However, how ROS signaling is coordinated with cell cycle progression and integrated into the cell cycle control machinery on the molecular level remains unsolved. Here, we report oscillations of mitochondrial ROS during the cell cycle that target cyclin-dependent kinase 2 (CDK2). Chemical and metabolic interference with ROS production decrease T-loop phosphorylation on CDK2, impeding its full activation and thus efficient DNA replication. ROS regulate CDK2 activity through oxidation of a conserved cysteine residue in close proximity to the T-loop, which prevents binding of the T-loop phosphatase KAP. Together our data reveal how ROS couple mitochondrial metabolism to DNA replication and cell cycle progression, and provide a solution to the longstanding conundrum of how KAP activity towards CDKs can be cell cycle-regulated.

cell biology↗

bioRxiv2020

UFMylation regulates translational homeostasis and cell cycle progression

UFMylation, the posttranslational modification of proteins with ubiquitin fold modifier 1 (UFM1) is essential for metazoan life and is associated with multiple human diseases. Although UFMylation has been linked to endoplasmic reticulum (ER) stress, its biological functions and relevant cellular targets beyond the ER are obscure. Here, we show that UFMylation directly controls translation and cell division in a manner otherwise known for cellular homeostasis-sensing pathways such as mTOR. By combining cell cycle analyses, mass spectrometry and ribosome profiling we demonstrate that UFMylation is required for eIF4F translation initiation complex assembly and recruitment of the ribosome. Interference with UFMylation shuts down global translation, which is sensed by cyclin D1 and halts the cell cycle independently of integrated stress response signalling. Our findings establish UFMylation as a key regulator of translation and uncover a pathway that couples translational homeostasis to cell cycle progression via a ubiquitin-like modification.

cell biology↗

bioRxiv2019

Redox potential defines functional states of adult hippocampal stem cells

Intracellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we show that in the hippocampus of adult mice it is the quiescent neural stem cells (NSCs) that maintain the highest ROS levels (hiROS). Classifying NSCs based on intracellular ROS content identified subpopulations with distinct molecular profiles, corresponding to functional states. Shifts in ROS content primed cells for a subsequent transition of cellular state, with lower cellular ROS content marking activity and differentiation. Physical activity, a known physiological activator of adult hippocampal neurogenesis, recruited the quiescent hiROS NSCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results describe a novel mechanism linking the modulation of cellular ROS by behavioral cues to the maintenance and activation of adult NSCs.\n\nHighlightsO_LIQuiescent adult hippocampal stem cells are characterized by high intracellular ROS\nC_LIO_LIChanges in intracellular ROS content precede changes in cellular state\nC_LIO_LIAcute physical activity recruits quiescent cells into active proliferation\nC_LIO_LIThis recruitment is marked by a Nox2-dependent ROS spike in hiROS stem cells and represents an independent mode of cell cycle entry\nC_LI\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/606186_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (65K):\norg.highwire.dtl.DTLVardef@1bbb7ddorg.highwire.dtl.DTLVardef@1241536org.highwire.dtl.DTLVardef@1ec9c32org.highwire.dtl.DTLVardef@102d527_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2018

An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules

Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracto. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models.

biochemistry↗

bioRxiv2018

Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

The conditional and reversible depletion of proteins by auxin-mediated degradation is a powerful tool to investigate protein functions in cells and whole organisms. However, its wider applications require fusing the auxin-inducible degron (AID) to individual target proteins. Thus, establishing the auxin system for multiple proteins can be challenging. Another approach for directed protein degradation are anti-GFP nanobodies, which can be applied to GFP stock collections that are readily available in different experimental models. Here, we combine the advantages of auxin and nanobody-based degradation technologies creating an AID-nanobody to degrade GFP-tagged proteins at different cellular structures in a conditional and reversible manner in human cells. We demonstrate efficient and reversible inactivation of the anaphase promoting complex/cyclosome (APC/C) and thus provide new means to study the functions of this essential ubiquitin E3 ligase. Further, we establish auxin degradation in a vertebrate model organism by employing AID-nanobodies in zebrafish.

cell biology↗

bioRxiv2017

Regulation of mitochondrial dynamics by Aurora A kinase

Aurora A kinase (AURKA) is a major regulator of mitosis and an important driver of cancer progression. The roles of AURKA outside of mitosis, and how these might contribute to cancer progression, are not well understood. Here we show that a fraction of cytoplasmic AURKA is associated with mitochondria, co-fractionating in cell extracts and interacting with mitochondrial proteins by reciprocal co-immunoprecipitation. We have also found that the dynamics of the mitochondrial network are sensitive to AURKA inhibition, depletion or overexpression. This can account for the different mitochondrial morphologies observed in RPE1 and U2OS cell lines, which show very different levels of expression of AURKA. We identify the mitochondrial fraction of AURKA as influencing mitochondrial morphology, since an N-terminally truncated version of the kinase that does not localize to mitochondria does not affect the mitochondrial network. We identify a cryptic mitochondrial targeting sequence in the AURKA N-terminus and discuss how alternative conformations of the protein may influence its cytoplasmic fate.

cell biology↗