Biology subjects

Shaul, Y.

Publications and source records attributed to Shaul, Y..

13 recordsLinked to original sources

bioRxiv2026

IKKα and IKKβ serve as kinase-independent scaffolds that prevent proteasomal degradation of NEMO

NF-{kappa}B essential modulator (NEMO) is the critical scaffold protein of the canonical I kappa B kinase (IKK) complex. Whilst studying the contribution of IKK and IKK{beta} to NF-{kappa}B signalling we noted that NEMO protein levels were reduced in IKK knockout (KO) HCT116 cells. Loss of NEMO was more pronounced in IKK KO cells than IKK{beta} KO, whilst IKK+IKK{beta} double knockout (DKO) cells exhibited a 90% reduction in NEMO abundance. In contrast, the abundance of IKKs was unaffected by NEMO KO, although this abolished canonical NF-{kappa}B activation. Loss of IKKs had no effect on NEMO mRNA levels but IKK DKO cells exhibited accelerated NEMO protein turnover that was reversed by the proteasome inhibitor, MG132, but not the NEDDylation inhibitor, MLN4924. TAK-243, a selective inhibitor of UBA1, the E1 ubiquitin-activating enzyme, increased basal NEMO levels in DKO cells but did not prevent NEMO turnover suggesting both ubiquitylation-dependent and independent pathways for degradation. Bioinformatic analysis identified substantial regions of intrinsic disorder within the N- and C-termini of NEMO. NEMO was rapidly degraded by isolated 20S proteasomes in vitro and this was inhibited by interaction with IKK or IKK{beta}. Finally, re-expression of IKK increased NEMO expression in IKK DKO cells and this required the C-terminal NEMO binding domain (NBD) but not a functional kinase domain. Thus the IKKs act as critical kinase-independent scaffolds that stabilise NEMO; loss of these interactions results in NEMO degradation via the proteasome. These results may be relevant to strategies for inhibiting NF-{kappa}B signalling by disrupting NEMO:IKK interactions.

biochemistry↗

bioRxiv2026

Genetic Information Processing Complexity as a Determinant of Virus Diversity

Viruses exhibit diverse genome architectures and replication strategies that shape their evolutionary trajectories and taxonomic diversification. Here, we test whether the complexity of viral genetic information processing predicts large-scale patterns of viral diversity. We define a propagation index that quantifies a minimal number of steps required for viral genome expression and replication across the Baltimore classes. Using ICTV taxonomy data (1971-2024), we identify a strong consistent linear relationship between the propagation index and viral diversification at both the family and genus levels. This statistically significant association is also observed for DNA and RNA viruses independently. Notably, the correlation persists across decades of ICTV releases despite substantial expansion and restructuring of viral taxonomy. Viruses with simpler propagation strategies consistently exhibit greater diversification, suggesting that genome processing complexity constrains macroevolutionary potential. These findings establish a quantitative link between propagation architecture and viral diversification and provide a predictive framework for understanding large-scale patterns of virus evolution.

evolutionary biology↗

bioRxiv2026

Disruption of the Brain-Spleen Axis Impairs Monocyte-Microglia Communication and Accelerates Disease Progression in a Model of Amyloidosis

Alzheimers disease (AD) is characterized by a prolonged asymptomatic phase before cognitive decline emerges, yet the mechanisms driving symptom onset remain unclear. Here, we hypothesized that the transition from asymptomatic to symptomatic disease is linked to dysfunction of brain-immune communication. Retrograde neuronal tracing in the 5xFAD mouse model of amyloidosis revealed reduced brain-spleen connectivity at advanced disease stages. To probe the functional role of the brain-spleen axis in coping with disease, we denervated the splenic nerve at an early presymptomatic stage. This intervention accelerated cognitive decline, impaired splenic hematopoiesis, diminished monocyte recruitment to the brain, disrupted monocyte-microglia signaling networks, and reduced the transition of microglia from a homeostatic to the disease-associated (DAM) state. Conversely, enhancing splenic noradrenergic input increased hematopoiesis, restored monocyte homing to the brain, and delayed cognitive impairment. The protective role of splenic monocytes was independently validated in a retinal cytotoxic injury model, in which splenic denervation impaired post-insult retinal ganglion cell survival. Together, these findings identify an active brain-spleen circuit in regulating monocyte recruitment and establish peripheral monocytes as key drivers of microglial state transitions and disease progression.

neuroscience↗

bioRxiv2025

Preliminary insights into salivary proteomic versus targeted biomarker profiles associated with acute physical fatigue

Introduction: Physical fatigue is a key determinant of operational readiness in tactical athletes. Hormonal, immune, and enzymatic biomarkers have been proposed for fatigue assessment, but reliability can be affected by external factors. Therefore, this study aimed to compare targeted stress-related biomarkers, proteins identified via untargeted salivary proteomics, and a combined panel of both for classifying acute physical fatigue. Methods: Ten recreationally active adults (6M, 4F) completed a fatiguing protocol within a 3-day sample collection window. Saliva samples were analyzed for targeted biomarkers via commercial immunoassays and proteins via untargeted liquid chromatography-mass spectrometry. Targeted, protein, and combined panels were identified using nested leave-one-subject-out cross-validation with FDR correction and stability selection, then classified via ridge-penalized logistic regression. Models were extended across the full protocol to evaluate classification of fatigue onset, rest, and recovery states. Results: A candidate identified protein (C1S) achieved slightly higher classification accuracy (80%) relative to a panel of targeted biomarkers (IgA and UA 70%), with superior sensitivity but similar specificity. In comparison, the combined panel, spanning immune (IgA), oxidative/metabolic (UA), complement (C1S), and cellular stress-related (ANG) outperformed both single-platform models with 90% accuracy, sensitivity, and specificity. Conclusions: Improved classification accuracy observed with the inclusion of candidate salivary proteins provide preliminary support for non-invasive proteomic monitoring of fatigue in tactical settings, and in particular the combination of complementary targeted and proteomic panels to capture a more complete physiological signature of acute fatigue. Future studies should validate these findings in larger, more diverse populations and assess applicability for chronic fatigue monitoring.

physiology↗

bioRxiv2025

c-Abl Phosphorylates Plk1 in Facilitating DNA Damage-Induced G2/M Checkpoint Release with a Trade-off of Micronuclei Formation

DNA double-strand breaks (DSBs) pose a critical threat to cellular proliferation and genomic integrity. Upon genotoxic stress, the DNA damage response (DDR) rapidly activates repair pathways and halts cell cycle progression through checkpoint activation. Previously, we demonstrated that DDR-activated c-Abl tyrosine kinase (ABL1) attenuates error-prone late-phase DSB repair. However, the broader functional implications of c-Abl in DDR regulation, and the fate of any residual unrepaired DNA fragments remained poorly understood. Here, we show that c-Abl regulates G2/M checkpoint release by targeting Polo-like kinase1 (Plk1). Depletion or inhibition of c-Abl resulted in increased G2-M accumulation and impaired checkpoint exit. We identified Y217 as a c-Abl phosphorylation site on Plk1, important for Plk1-mediated Claspin destabilization, a key step in G2/M checkpoint release. CRISPR-mediated introduction of the phopsho-silencing Plk1 Y217F mutation or the phospho-mimicking Y217E mutation into cells resulted in impaired and enhanced G2/M checkpoint release, respectively. Intriguingly, c-Abl-mediated G2/M checkpoint release correlated with elevated DNA damage-induced micronuclei (MNi) formation. Depletion or inhibition of c-Abl reduced MNi formation, whereas induction of c-Abl expression increased it, implicating c-Abl as an active effector in both processes. We propose a trade-off model whereby, following the rapid initial repair phase, c-Abl shifts cellular priorities from prolonged, potentially error-prone DSB repair toward cell cycle resumption, thereby promoting G2-M checkpoint exit and DDR deactivation at the cost of increased MNi formation. Our findings describe a novel regulatory DDR axis involving c-Abl and Plk1 and provide mechanistic insights into how DDR termination is orchestrated.

biochemistry↗

bioRxiv2025

Proteasome Caspase-Like Activity Regulates Stress Granules and Proteasome Condensates

The 20S proteasome maintains cellular protein homeostasis, particularly during stress responses. In a previous study, we identified numerous 20S proteasome substrates through mass spectrometry analysis of peptides generated from cellular extracts degraded by purified 20S proteasome. Many substrates were found to be components of liquid-phase separation, such as stress granules (SGs). Here, we demonstrate the degradation products predominantly arise from the caspase-like (CL) proteasomal activity. To investigate the functional implications of CL activity, we generated cell lines devoid of CL function by introducing the PSMB6 T35A mutation. These mutant cells exhibited slower growth rates, heightened sensitivity to stress, and activation of the unfolded protein response (UPR), as indicated by elevated levels of spliced XBP1 (sXBP1) and stress markers. Cells were subjected to arsenite and osmotic stress to assess their responses. Our findings reveal that CL activity is crucial for efficient SG assembly but does not significantly affect SG disassembly. Interestingly, in these mutant cells, proteasomes were more cytoplasmic under normal conditions but formed nuclear condensates/granules (PGs) upon NaCl osmotic stress. However, the PGs were unstable and rapidly disassembled. These findings underscore the important role of the proteasomes CL activity in managing stress-induced dynamics of liquid-liquid phase, highlighting its importance in cellular adaptation to proteotoxic and genotoxic stress conditions.

cell biology↗

bioRxiv2024

Investigating the p21 Ubiquitin-Independent Degron Reveals a Dual Degron Module Regulating p21 Degradation and Function.

A group of intrinsically disordered proteins (IDPs) are subjected to 20S proteasomal degradation in a ubiquitin-independent manner. Recently, we have reported that many IDP/IDR are targeted to the 20S proteasome via interaction with the C-terminus of PSMA3 subunit, termed the PSMA3 Trapper. In this study, we investigated the biological significance of the IDP-Trapper interaction using the IDP p21. Using split luciferase reporter assay and conducting detailed p21 mutagenesis, we first identified the p21 RRLIF box, localized at the C-terminus, as mediating the Trapper interaction in cells. To demonstrate the role of this box in p21 degradation, we edited the genome of HEK293 and HeLa cell lines using a CRISPR strategy. We found that the p21 half-life increased in cells with either a deleted or mutated p21 RRLIF box. The edited cell lines displayed an aberrant cell cycle pattern under normal conditions and in response to DNA damage. Remarkably, these cells highly expressed senescence hallmark genes in response to DNA damage, highlighting that the increased p21 half-life, not its actual level, regulates senescence. Our findings suggest that the p21 RRLIF box, which mediates interaction with the PSMA3 Trapper, acts as a ubiquitin-independent degron. This degron is positioned adjacent to the previously identified ubiquitin-dependent degron, forming a dual degron module that functionally regulates p21 degradation and its physiological outcomes.

molecular biology↗

bioRxiv2023

The TMPRSS2 non-protease domains regulating SARS-CoV-2 Spike in mediated virus entry

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Through the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus infection, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2s cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.

microbiology↗

bioRxiv2018

Recruitment of the protein phosphatase-1 catalytic subunit to promoters by the dual-function transcription factor RFX1

SummeryRFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes.\n\nThe exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes.

molecular biology↗

bioRxiv2018

A short HBV RNA region induces RNR-R2 expression in non-cycling cells and in primary human hepatocytes

Hepatitis B virus infects non-dividing cells in which dNTPs are scarce. HBV replication requires dNTPs. To cope with this constraint the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels. Previously we reported that the HBx open reading frame (ORF) triggers this pathway. Unexpectedly however, we report here that the production of HBx protein is not essential. We found that a small region of 125 bases within the HBx transcript is sufficient to induce RNR-R2 expression in growth arrested HepG2 cells and in primary human hepatocytes (PHH). The observed HBx embedded regulatory element is named ERE. We demonstrate that ERE is functional as a positive strand RNA polymerase-II transcript. Remarkably, ERE is sufficient to induce the Chk1-E2F1-RNR-R2 DDR pathway, previously reported to be activated by HBV. Furthermore, we found that ERE activates ATR but not ATM in eliciting this DDR pathway in upregulating RNR-R2. These findings demonstrate the multitasking role of HBV transcripts in mediating virus-host cell interaction, a mechanism that explains how such a small genome effectively serves such a pervasive virus.\n\nAuthor summaryThe hepatitis B virus (HBV) infects the human liver and over 250 million people worldwide are chronically infected with HBV and at risk for cirrhosis and liver cancer. HBV has a very small DNA genome with only four genes, much fewer than other viruses. For propagation the virus consumes dNTPs, the building blocks of DNA, in much higher amounts than the infected cells provide. To cope with this constraint, the virus manipulates the cells to increase the production of dNTPs. We found that the virus activates the cellular response to DNA damage upon which the cells increase the production of dNTPs, but instead of repairing cellular DNA, the virus uses them for production of its own DNA. Usually viruses manipulate host cells with one or more of their unique proteins, however the small HBV genome cannot afford having such a unique gene and protein. Instead, we found that here the virus relies on RNA to manipulate the host cells. Our findings highlight the unprecedented principle of a multitasking viral RNA that is not only designed to be translated into proteins but also harbors an independent role in activating the cellular DNA damage response.

microbiology↗

bioRxiv2017

Oncogenic addiction to high 26S proteasome levels

Proteasomes are large intracellular complexes responsible for the degradation of cellular proteins. The altered protein homeostasis of cancer cells results in increased dependency on proteasome function. There are several different proteasome complexes that may be assembled in cells, with the 20S catalytic core common to them all. 20S proteasomes can function in isolation, or as part of larger complexes (26S) with regulatory particles (RP) such as the 19S that is needed for the targeting and processing of ubiquitinated substrates. Proteasome inhibitors target the catalytic barrel (20S) and thus this inhibition does not allow the deconvolution of the distinct roles of 20S vs. 26S proteasomes in cancer progression. We examined the degree of dependency of cancer cells specifically to the level of the 26S proteasome complex. We found that oncogenic transformation of human and mouse immortalized cells with mutant Ras induced a strong increase in the translation of the 26S proteasome subunits, giving rise to high 26S complex levels. We show that depletion of a single subunit of the 19S RP was sufficient to significantly reduce the 26S proteasome level and lower the cellular 26S/20S ratio. We further demonstrate that the accumulated 26S proteasome was essential for the viability of the transformed cells. Moreover, the viability of 20 different cancer cell lines, but not normal human fibroblasts, was severely compromised upon specific 26S proteasome suppression regardless of their p53 status. Suppression of 26S activated the UPR and Caspase-3, which at least partially explains the cell-killing effect. Morphologically, suppression of the 26S proteasome resulted in cytoplasm shrinkage and nuclear deformation. Thus, the tumor cell-specific addiction to high 26S proteasome levels sets the stage for future strategies in cancer therapy.

cancer biology↗

bioRxiv2017

A 20S proteasome receptor for degradation of intrinsically disordered proteins

Degradation of intrinsically disordered proteins (IDPs) by the 20S proteasome, unlike ubiquitin-dependent 26S proteasomal degradation, does not require proteasomal targeting by polyubiquitin. However, how these proteins are recognized by the proteasome was unknown. We report here on a mechanism of 20S proteasome targeting. Analysis of protein interactome datasets revealed that the proteasome subunit PSMA3 interacts with many IDPs. By employing in vivo and cell-free experiments we demonstrated that the PSMA3 C-terminus binds p21, c-Fos and p53, all IDPs and 20S proteasome substrates. A 69 amino-acids long fragment is autonomously functional in interacting with IDP substrates. Remarkably, this fragment in isolation blocks the degradation of a large number of IDPs in vitro and increases the half-life of proteins in vivo. We propose a model whereby the PSMA3 C-terminal region plays a role of substrate receptor in the process of proteasomal degradation of many IDPs.

molecular biology↗

bioRxiv2017

NQO1 Binds And Supports SIRT1 Function

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

cell biology↗