Biology subjects

Ramadan, K.

Publications and source records attributed to Ramadan, K..

10 recordsLinked to original sources

bioRxiv2026

DNA-induced conformational changes in SPRTN relieve its auto-inhibitory effect on protease activity

The DNA-dependent metalloprotease SPRTN has emerged as a key enzyme in the proteolysis of DNA-protein crosslinks (DPCs), thereby protecting us against genome instability, accelerated ageing, and cancer. DNA and ubiquitin chains serve as the primary activator and catalyst of SPRTN proteolysis, respectively, but how they promote SPRTN activation and activity remains incompletely understood. To address this question, we developed a highly sensitive multi-turnover fluorescence resonance energy transfer (FRET) assay to monitor SPRTN proteolysis in real time. We found that the auto-cleaved N-terminal SPRTN fragment, comprising the metalloprotease domain (MPD), zinc-binding domain (ZBD), and basic region (BR), is highly stable, enzymatically active, and retains ubiquitin-dependent activation. Interestingly, the MPD alone exhibits basal intrinsic activity that is independent of both DNA activation and ubiquitin avidity effect. We show that ZBD and MPD together exert steric regulation: ZBD maintains MPD in an autoinhibited state, while MPD largely prevents ZBD from binding DNA. BR, together with DNA, is essential to relieve ZBD-mediated inhibition of MPD. Using a site-trapping approach, we demonstrate that the ZBD-BR-DNA trinity induces an open conformation of the SPRTN N-terminus in cis, thereby releasing autoinhibition. MPD and BR together restrict the DNA-binding stoichiometry of ZBD, enabling SPRTN to function efficiently in proximity to DNA despite its low abundance in vivo. Collectively, our work overturns the long-standing dogma that SPRTN autocleavage inactivates the enzyme and reveals how DNA-induced conformational changes in SPRTN fine-tune its protease activity, providing a prerequisite for subsequent ubiquitin activation and rapid proteolysis of DPCs.

biochemistry↗

bioRxiv2025

Nucleophagy removes cytotoxic trapped PARP1

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) induce cytotoxicity in homologous recombination repair (HRR)-deficient cancers by causing PARP1 to become trapped on chromatin, resulting in irreparable replication-associated DNA damage. Although increased clearance of trapped PARP1 from chromatin reduces the sensitivity of cancer cells to PARPi, details surrounding this process remain unclear. PARPi exposure is known to cause increased autophagy flux, whilst autophagy inhibition can hypersensitise cells to PARPi. Using various biochemical, cell biological and live imaging-based assays, we found that trapped PARP1 is cleared by nucleophagy, the selective autophagy of nuclear substrates. Specifically, the nucleophagy of trapped PARP1 was orchestrated by the selective autophagy receptor TEX264 and its partner segregase p97/VCP. TEX264 mediates this process by directly interacting with trapped PARP1, thus bridging PARP1 to the autophagosomal resident protein LC3 for processing via autophagy. Impeding this process, either chemically or genetically, heightened PARP1 trapping, leading to accumulation of protein aggregates, replication-associated DNA damage and cell lethality, re-sensitising PARPi-resistant cells to various PARPi. In conclusion, we show that nucleophagy acts in a cytoprotective manner to directly target PARPi-induced trapped PARP1 for degradation.

cell biology↗

bioRxiv2025

Degeneration and Impaired Resilience of Skull Bone and Hematopoietic Bone Marrow

Bone marrow health is central to transplantations, blood formation, and cancer progression. However, the bone marrow niche deteriorates with age, impairing haematopoietic stem cell function. Contrary to a recent report1 suggesting skull marrow resists ageing, our multi-laboratory investigation reveals the opposite: the skull marrow is among the vulnerable sites of age-related decline. Ageing skull niches consistently show loss of mesenchymal and osteoprogenitors, suppression of angiogenic and lymphatic programs, adipocyte accumulation, vascular senescence, DNA replication stress, mitochondrial dysfunction, cellular senescence, and heightened inflammation. Proteomic profiling further highlights this vulnerability, demonstrating that vertebral niches--unlike the skull--are relatively spared from these ageing hallmarks. Together, these convergent datasets overturn the notion of skull-specific resilience and instead establish the skull marrow as a fragile, degenerating environment. These findings redefine marrow ageing and highlight the skull as a critical, clinically relevant target for sustaining blood and immune health and reducing vulnerability to haematological disease.

cell biology↗

bioRxiv2024

The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks

DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognises its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs still needs to be discovered. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain named the Ubiquitin interface of SprT Domain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains. SPRTN binding to ubiquitin chains via USD leads to [~] 67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. This study reveals the ubiquitination of DPCs is the key signal for SPRTNs substrate specificity and rapid proteolysis.

biochemistry↗

bioRxiv2022

LARP1 regulates metabolism and mTORC1 activity in cancer

The protein mammalian target of rapamycin (mTOR) is a master regulator of cell homeostasis. Although mTOR is aberrantly overactivated in 70% ovarian cancers, mTOR cascade inhibitors (such as those blocking the kinase activity of mTOR itself or upstream kinases PI3K/AKT) have demonstrated disappointing activity in ovarian cancer clinical trials. These findings indicate that, despite its pivotal role in normal cells, hyperactivated mTOR does not act as a master regulator of metabolism in this cancer context. Surprisingly, we have identified that the RNA binding protein LARP1, a known phospho-target of mTORC1 and activator of ribosomal biogenesis, is responsible for metabolic reprogramming in mTOR-dysregulated cancers. LARP1 post-transcriptionally regulates the expression of several hundred rate-limiting enzymes involved in multiple aspects of metabolism, including glycolysis and oxidative phosphorylation. Through this mechanism LARP1 sustains ATP production and mTORC1 localisation on the lysosome, thereby activating cell proliferation despite the scarcity of extracellular nutrients. Our findings show that, by sustaining global cellular metabolism in response to growth factor signalling, LARP1 has a central post-transcriptional role in controlling mTORC1 localisation and driving cancer progression, a key cancer hallmark.

cancer biology↗

bioRxiv2021

The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin

Summary paragraphPoly-(ADP-ribose) polymerase inhibitors (PARPi) elicit anti-tumour activity in homologous recombination defective cancers by promoting cytotoxic, chromatin-bound, "trapped" PARP1. How cells process trapped PARP1 remains unclear. By exploiting wild-type or trapping-resistant PARP1 transgenes combined with either a rapid immunoprecipitation mass-spectrometry of endogenous proteins (RIME)-based approach, or PARP1 Apex2-proximity labelling linked to mass-spectrometry, we generated proteomic profiles of trapped and non-trapped PARP1 complexes. This combined approach identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase (aka VCP). Subsequent experiments demonstrated that upon trapping, PARP1 is SUMOylated by the SUMO-ligase PIAS4 and subsequently ubiquitinated by the SUMO-targeted E3-ubiquitin ligase, RNF4, events that promote p97 recruitment and p97 ATPase-mediated removal of trapped-PARP1 from chromatin. Consistent with this, small molecule p97 complex inhibitors, including a metabolite of the clinically-used drug disulfiram (CuET) that acts as a p97 sequestration agent, prolong PARP1 trapping and thus enhance PARPi-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Taken together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 from chromatin and the response of homologous recombination defective tumour cells to PARPi.

molecular biology↗

bioRxiv2021

The NUCKS1-SKP2-p21/p27 axis controls S phase entry

Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. TCGA data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.

cancer biology↗

bioRxiv2020

SPRTN protease and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

DNA-protein crosslinks (DPCs) are a specific type of DNA lesions where proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration and accelerated ageing. DPC proteolysis was recently discovered as a specialised pathway for DPC repair. The DNA-dependent SPRTN protease and 26S proteasome emerged as as two independent proteolytic systems for DPC repair. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the role of ubiquitin and SUMO in DPC repair, we identified mutually exclusive signalling mechanisms associated with DPC repair pathway choice. DPC modification by SUMO-1 favours SPRTN proteolysis as the preferred pathway for DPC repair. DPC SUMOylation counteracts DPC ubiquitination, which promotes DNA breaks and the switch to HR. We propose that modification of DPCs by SUMO-1 promotes SPRTN proteolysis, which is essential for DPC removal to prevent DNA replication defects, chromosomal recombination and genomic instability.

cell biology↗

bioRxiv2019

The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

cell biology↗

bioRxiv2018

SPRTN Protease and Checkpoint Kinase 1 Cross-Activation Loop Safeguards DNA Replication

The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease activity exhibit severe DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer in humans and mice. Strikingly, SPRTN-deficient cells also show a severe G2/M checkpoint defect and fail to activate a robust checkpoint kinase 1 (CHK1) signalling cascade normally triggered in response to replication stress. Here, we show that SPRTN activates the CHK1 signalling cascade during physiological (steady-state) DNA replication by proteolysis-dependent eviction of CHK1 from chromatin. The N-terminal CHK1 fragments cleaved by SPRTN protease still possess kinase activity and phosphorylate SPRTN. This CHK1-dependent phosphorylation stimulates SPRTN recruitment to chromatin to further promote CHK1 eviction from chromatin, DPC removal and unperturbed DNA replication. Our data suggest that a SPRTN-CHK1 cross-activation loop is essential for steady-state DNA replication and protection from DNA replication stress, a major source of genome instability in diseases that cause premature ageing and cancer. In addition, we disclose a mechanism of CHK1 activation during physiological DNA replication, when long stretches of single stranded (ss) DNA that are induced by genotoxic stress and activate canonical and robust CHK1 signalling are not present.

cell biology↗