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International Journal of Biological... Jun 2024The coordination of enzymes and regulatory proteins for eukaryotic DNA replication and repair is largely achieved by Proliferating Cell Nuclear Antigen (PCNA), a...
The coordination of enzymes and regulatory proteins for eukaryotic DNA replication and repair is largely achieved by Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimeric protein that embraces the DNA duplex. Many proteins bind PCNA through a conserved sequence known as the PCNA interacting protein motif (PIP). PCNA is further regulated by different post-translational modifications. Phosphorylation at residue Y211 facilitates unlocking stalled replication forks to bypass DNA damage repair processes but increasing nucleotide misincorporation. We explore here how phosphorylation at Y211 affects PCNA recognition of the canonical PIP sequences of the regulatory proteins p21 and p15, which bind with nM and μM affinity, respectively. For that purpose, we have prepared PCNA with p-carboxymethyl-L-phenylalanine (pCMF, a mimetic of phosphorylated tyrosine) at position 211. We have also characterized PCNA binding to the non-canonical PIP sequence of the catalytic subunit of DNA polymerase δ (p125), and to the canonical PIP sequence of the enzyme ubiquitin specific peptidase 29 (USP29) which deubiquitinates PCNA. Our results show that Tyr211 phosphorylation has little effect on the molecular recognition of p21 and p15, and that the PIP sequences of p125 and USP29 bind to the same site on PCNA as other PIP sequences, but with very low affinity.
PubMed: 38880460
DOI: 10.1016/j.ijbiomac.2024.133187 -
Cancer Letters Jun 2024Deubiquitylases (DUBs) have emerged as promising targets for cancer therapy due to their role in stabilizing substrate proteins within the ubiquitin machinery. Here, we...
Deubiquitylases (DUBs) have emerged as promising targets for cancer therapy due to their role in stabilizing substrate proteins within the ubiquitin machinery. Here, we identified ubiquitin-specific protease 26 (USP26) as an oncogene via screening prognostic DUBs in breast cancer. Through in vitro and in vivo experiments, we found that depletion of USP26 inhibited breast cancer cell proliferation and invasion, and suppressed tumor growth and metastasis in nude mice. Further investigation identified co-chaperone Bcl-2-associated athanogene 3 (BAG3) as the direct substrate of USP26, and ectopic expression of BAG3 partially reversed antitumor effect induced by USP26 knockdown. Mechanistically, the lysine acetyltransferase Tip60 targeted USP26 at K134 for acetylation, which enhanced USP26 binding affinity to BAG3, leading to BAG3 deubiquitination and increased protein stability. Importantly, we employed a structure-based virtual screening and discovered a drug-like molecule called 5813669 that targets USP26, destabilizing BAG3 and effectively mitigating tumor growth and metastasis in vivo. Clinically, high expression levels of USP26 were correlated with elevated BAG3 levels and poor prognosis in breast cancer patients. Overall, our findings highlight the critical role of USP26 in BAG3 protein stabilization and provide a promising therapeutic target for breast cancer.
PubMed: 38880224
DOI: 10.1016/j.canlet.2024.217005 -
Cell Reports Jun 2024p53 regulates multiple signaling pathways and maintains cell homeostasis under conditions of DNA damage and oxidative stress. Although USP7 has been shown to promote p53...
p53 regulates multiple signaling pathways and maintains cell homeostasis under conditions of DNA damage and oxidative stress. Although USP7 has been shown to promote p53 stability via deubiquitination, the USP7-p53 activation mechanism has remained unclear. Here, we propose that DNA damage induces reactive oxygen species (ROS) production and activates ATM-CHK2, and CHK2 then phosphorylates USP7 at S168 and T231. USP7 phosphorylation is essential for its deubiquitination activity toward p53. USP7 also deubiquitinates CHK2 at K119 and K131, increasing CHK2 stability and creating a positive feedback loop between CHK2 and USP7. Compared to peri-tumor tissues, thyroid cancer and colon cancer tissues show higher CHK2 and phosphorylated USP7 (S168, T231) levels, and these levels are positively correlated. Collectively, our results uncover a phosphorylation-deubiquitination positive feedback loop involving the CHK2-USP7 axis that supports the stabilization of p53 and the maintenance of cell homeostasis.
Topics: Checkpoint Kinase 2; Ubiquitin-Specific Peptidase 7; Humans; Tumor Suppressor Protein p53; Phosphorylation; Ubiquitination; Oxidative Stress; Feedback, Physiological; DNA Damage; Reactive Oxygen Species; Ataxia Telangiectasia Mutated Proteins; Signal Transduction; Cell Line, Tumor; Protein Stability; Animals
PubMed: 38879877
DOI: 10.1016/j.celrep.2024.114366 -
Journal of Experimental Botany Jun 2024Light serves as a pivotal environmental cue regulating various aspects of plant growth and development, including seed germination, seedling de-etiolation, and shade...
Light serves as a pivotal environmental cue regulating various aspects of plant growth and development, including seed germination, seedling de-etiolation, and shade avoidance. Within this regulatory framework, the basic helix-loop-helix transcription factors known as PHYTOCHROME INTERACTING FACTORS (PIFs) play an essential role in orchestrating responses to light stimuli. Phytochromes, acting as red/far-red light receptors, initiate a cascade leading to the degradation of PIFs (except PIF7), thereby triggering transcriptional reprogramming to facilitate photomorphogenesis. Recent research has unveiled multiple post-translational modifications that regulate the abundance and/or activity of PIFs, including phosphorylation, dephosphorylation, ubiquitination, deubiquitination and SUMOylation. Moreover, intriguing findings indicate that PIFs can influence chromatin modifications. These include modulation of Histone 3 Lysine-9 acetylation (H3K9ac), as well as occupancy of histone variants such as H2A.Z (associated with gene repression) and H3.3 (associated with gene activation), thereby intricately regulating downstream gene expression in response to environmental cues. This review summarizes recent advances in understanding PIFs' role in regulating various signaling pathways with a major focus on photomorphogenesis.
PubMed: 38877836
DOI: 10.1093/jxb/erae276 -
The Journal of Biological Chemistry Jun 2024Chemotherapeutic agents for treating colorectal cancer primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system (UPS) is critical for apoptosis...
Chemotherapeutic agents for treating colorectal cancer primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system (UPS) is critical for apoptosis regulation. Deubiquitinating enzymes (DUBs) remove ubiquitin from substrates to reverse ubiquitination. Although over 100 DUB members have been discovered, the biological functions of only a small proportion of DUBs have been characterized. Here, we aimed to systematically identify the DUBs that contribute to the development of colorectal cancer. Among the DUBs, ubiquitin-specific protease 36 (USP36), is upregulated in colorectal cancer. We showed that the knockdown of USP36 induces intrinsic and extrinsic apoptosis. Through gene silencing and coimmunoprecipitation techniques, we identified survivin and cIAP1 as USP36 targets. Mechanistically, USP36 binds and removes lysine-11 (K11)-linked ubiquitin chains from cIAP1 and lysine-48 (K48)-linked ubiquitin chains from survivin to abolish protein degradation. Overexpression of USP36 disrupts the formation of the XIAP-Smac complex and promotes RIPK1 ubiquitination, validating USP36 as an inhibitor to intrinsic and extrinsic apoptosis through deubiquitinating survivin and cIAP1. Therefore, our results suggest that USP36 is involved in colorectal cancer progression and is a potential therapeutic target.
PubMed: 38876304
DOI: 10.1016/j.jbc.2024.107463 -
Neoplasia (New York, N.Y.) Jun 2024Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by...
Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by EBV, is a lytic cycle protein with deubiquitinating activity has been found to participate in disrupting repair of DNA damage. We first confirmed that BPLF1 gene in gastric cancer (GC) significantly increased the DNA double strand breaks (DSBs). Ubiquitination mass spectrometry identified histones as BPLF1 interactors and potential substrates, and co-immunoprecipitation and in vitro experiments verified that BPLF1 regulates H2Bub by targeting Rad6. Over-expressing Rad6 restored H2Bub but partially reduced γ-H2AX, suggesting that other downstream DNA repair processes were affected. mRNA expression of BRCA2 were significantly down-regulated by next-generation sequencing after over-expression of BPLF1, and over-expression of p65 facilitated the repair of DSBs. We demonstrated BPLF1 may lead to the accumulation of DSBs by two pathways, reducing H2B ubiquitination (H2Bub) and blocking homologous recombination which may provide new ideas for the treatment of gastric cancer.
PubMed: 38875930
DOI: 10.1016/j.neo.2024.101012 -
Brain : a Journal of Neurology Jun 2024USP25 encodes ubiquitin-specific proteases 25, a key member of deubiquitinating enzyme family and is involved in neural fate determination. Although abnormal expression...
USP25 encodes ubiquitin-specific proteases 25, a key member of deubiquitinating enzyme family and is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown etiology. Five heterozygous USP25 variants including two de novo and three co-segregated variants were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared to the East Asian population and all populations in the gnomAD database. The mean onset ages of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom except one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was ubiquitously expressed in mouse brain with two peaks on embryonic days (E14‒E16) and postnatal day 21, respectively. Similarly, USP25 expressed in fetus/early childhood stage with a second peak at approximately 12‒20 years old in human brain, consistent with the seizure onset age at infancy and juvenile in the patients. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knock-out mice, which showed increased seizure susceptibility compared to wild-type mice in pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we employed multiple functional detections. In HEK293T cells, the severe phenotype associated variant (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed a stable truncated dimers with increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del increased neuronal excitability in mice brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.
PubMed: 38875478
DOI: 10.1093/brain/awae191 -
Renal Failure Dec 2024As a pattern recognition receptor, Toll-like receptor 4 (TLR4) is crucial for the development and progression of acute kidney injury (AKI). This study aims to explore...
Usp9x contributes to the development of sepsis-induced acute kidney injury by promoting inflammation and apoptosis in renal tubular epithelial cells via activation of the TLR4/nf-κb pathway.
As a pattern recognition receptor, Toll-like receptor 4 (TLR4) is crucial for the development and progression of acute kidney injury (AKI). This study aims to explore whether the deubiquitinase Usp9x influences the TLR4/NF-B pathway to cause sepsis-induced acute kidney injury (S-AKI). The model of AKI was established in Sprague-Dawley rats using the cecal ligation and puncture (CLP) method, while renal tubular epithelial cell NRK-52E was stimulated with lipopolysaccharide (LPS) . All plasmids were transfected into NRK-52E cells according to the indicated group. The deubiquitinase of TLR4 was predicted by the online prediction software Ubibrowser. Subsequently, Western blot and Pearson correlation analysis identified Usp9x protein as a potential candidate. Co-IP analysis verified the interaction between TLR4 and Usp9x. Further research revealed that overexpression of Usp9x inhibited degradation of TLR4 protein by downregulating its ubiquitination modification levels. Both and experiments observed that interference with Usp9x effectively alleviated the inflammatory response and apoptosis of renal tubular epithelial cells (RTECs) induced by CLP or LPS, whereas overexpression of TLR4 reversed this situation. Transfection with sh-Usp9x in NRK-52E cells suppressed the expression of proteins associated with the TLR4/NF-κB pathway induced by LPS. Moreover, the overexpression of TLR4 reversed the effect of sh-Usp9x transfection. Therefore, the deubiquitinase Usp9x interacts with TLR4, leading to the upregulation of its expression through deubiquitination modification, and the activation of the TLR4/NF-κB signaling pathway, thereby promoting inflammation and apoptosis in renal tubular epithelial cells and contributing to sepsis-induced acute kidney injury.
Topics: Animals; Toll-Like Receptor 4; Acute Kidney Injury; Sepsis; Apoptosis; Rats, Sprague-Dawley; NF-kappa B; Rats; Epithelial Cells; Signal Transduction; Kidney Tubules; Ubiquitin Thiolesterase; Male; Inflammation; Disease Models, Animal; Cell Line; Lipopolysaccharides; Ubiquitination
PubMed: 38874156
DOI: 10.1080/0886022X.2024.2361089 -
Journal of Medicinal Chemistry Jun 2024Papain-like protease (PLpro) is a promising therapeutic target for its pivotal role in the life cycle of SARS-CoV-2. A series of 1,2,4-oxadiazole derivatives was...
Design, Synthesis, and Biological Evaluation of 1,2,4-Oxadiazole Derivatives Containing an Aryl Carboxylic Acid Moiety as Potent Sarbecovirus Papain-like Protease Inhibitors.
Papain-like protease (PLpro) is a promising therapeutic target for its pivotal role in the life cycle of SARS-CoV-2. A series of 1,2,4-oxadiazole derivatives was designed and synthesized via a ring formation strategy based on SARS-CoV-2 PLpro-GRL0617 complex structure. Systematic structure-activity relationship studies revealed that introducing oxadiazole and aryl carboxylic acid moieties to GRL0617 enhanced the enzymatic inhibition activity, affinity, and deubiquitination capacity toward PLpro. 1,2,4-Oxadiazole compounds and , which had PLpro inhibition activity (IC = 1.8 and 1.0 μM) and antiviral activity against SARS-CoV-2 (EC = 5.4 and 4.3 μM), exhibited good metabolic stability ( > 93.2 min) and higher plasma exposure (AUC = 17,380.08 and 24,289.76 ng·h/mL) in mice. Especially, compound with moderate oral bioavailability of 39.1% and potent antiviral activity is worthy of further studies . Our findings provide a new insight for the discovery of antiviral agents targeting PLpro.
Topics: Oxadiazoles; Drug Design; Animals; Antiviral Agents; Structure-Activity Relationship; SARS-CoV-2; Mice; Humans; Carboxylic Acids; Molecular Docking Simulation; Protease Inhibitors; COVID-19 Drug Treatment; Coronavirus Papain-Like Proteases
PubMed: 38871484
DOI: 10.1021/acs.jmedchem.4c00534 -
Access Microbiology 2024KSHV viral FLICE inhibitory protein (vFLIP) is a potent activator of NF-κB signalling and an inhibitor of apoptosis and autophagy. Inhibition of vFLIP function and...
KSHV viral FLICE inhibitory protein (vFLIP) is a potent activator of NF-κB signalling and an inhibitor of apoptosis and autophagy. Inhibition of vFLIP function and NF-κB signalling promotes lytic reactivation. Here we provide evidence for a novel function of vFLIP through inhibition of the deubiquitinating (DUB) activity of the negative regulator, A20. We demonstrate direct interaction of vFLIP with Itch and A20 and provide evidence for subsequent loss of A20 DUB activity. Our results provide further insight into the function of vFLIP in the regulation of NF-κB signalling.
PubMed: 38868372
DOI: 10.1099/acmi.0.000625.v4