Macrocyclic Peptide-Based Dual-Sensor Platform for Linkage-Specific Visualization of Ubiquitin Chain Assembling in Live Cells

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Янв. 21, 2025

Intracellular monitoring of protein ubiquitination and differentiating polyubiquitin chain topology are crucial for understanding life processes drug discovery, which is challenged by the high complexity process a lack molecular tools. Herein, synthetic dual-sensor platform specific K48-linked ubiquitin oligomers was tailored in situ visualization assembling live biosystems. This achieved using macrocyclic peptides as recognition motifs tetraphenylethylene derivative an activatable reporter. The efficient cell penetration, tight binding, protection delivered "freeze-and-image" approach, allowing fluorescent readout linkage type elongation without perturbing physiological environment. Motivated these unique features, mapping K48-ubiquitination dynamics during degradation facilely achieved. Rapid, sensitive, intracellular assessment mechanism action potency dependence proteolysis-targeting chimeras (PROTACs) demonstrated, presenting sensors promising tools PROTAC development.

Язык: Английский

---

A bacterial RING ubiquitin ligase triggering stepwise degradation of BRISC via TOLLIP-mediated selective autophagy manipulates host inflammatory response

Autophagy, Год журнала: 2025, Номер unknown, С. 1 - 20

Опубликована: Фев. 27, 2025

Numerous bacterial pathogens have evolved tactics to interfere with the host ubiquitination network evade clearance by innate immune system. Nevertheless, subtle antagonism between a ubiquitinase and deubiquitinase, through which they modify their respective targets within multifaceted network, has yet be characterized. BRCC3 isopeptidase complex (BRISC) is newly identified K63-specific deubiquitinase that plays crucial role in cellular signaling pathways such as inflammation. NleG, type III secretion system (T3SS) effector, contains conserved RING E3 ubiquitin ligase domain interacts machinery, along distinct substrate-recognition proteins. Here, one particular variant, NleG6, was mediating K27- K29-linked polyubiquitination at residues K89 K114 of ABRAXAS2/FAM175B, scaffolding protein BRISC complex, leading its degradation TOLLIP (toll interacting protein)-mediated selective autophagy. Further investigations elucidated ABRAXAS2 triggered subsequent adjacent BRCC3, turn, hindered TNIP1/ABIN1 degradation, ultimately inhibiting NFKB/NF-κB (nuclear factor kappa B)-mediated inflammatory responses. This chain events offers valuable insights into NFKB activation deubiquitinating BRISC, unveiling how bacteria manipulate regulation autophagy inhibit host's response thus dominate pathogen-host tug-of-war.Abbreviations: 3-MA: 3-methyladenine; A/E: attaching effacing; ATG7: related 7; BafA1: bafilomycin A1; BNIP3L/Nix: BCL2 3 like; BRISC: complex; Cas9: CRISPR-associated 9; co-IP: co-immunoprecipitation; CQ: chloroquine; CRISPR: clustered regulatory interspaced short palindromic repeat; DAPI: 4',6-diamidino2-phenylindole; DMSO: dimethyl sulfoxide; DUB: enzyme; E. coli: Escherichia coli; EHEC: enterohemorrhagic EPEC: enteropathogenic GFP: green fluorescent protein; LEE: locus enterocyte effacement; MAP1LC3B/LC3: microtubule associated 1 light beta; MG132: cbz-leu-leu-leucinal; MOI: multiplicity infection; NBR1: NBR1 cargo receptor; NC: negative control; NFKB/NF-κB: nuclear B; NH4Cl: ammonium chloride; OPTN: optineurin; SQSTM1/p62: sequestosome 1; sgRNAs: small guide RNAs; T3SS: system; TNF: tumor necrosis factor; TOLLIP: toll TRAF: TNF receptor TUBB: tubulin beta class I; WCL: whole cell lysate; WT: wide type.

Язык: Английский

---

USP20 mitigates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing HuR

Research Square (Research Square), Год журнала: 2025, Номер unknown

Опубликована: Апрель 16, 2025

Background The severe cardiotoxicity of doxorubicin (Dox) significantly restricts its clinical application. Deubiquitinating enzymes (DUBs) play pivotal roles in cardiac pathophysiology because their precise regulation protein function, localization and degradation.Objectives objective this study was to investigate the role molecular mechanism ubiquitin-specific peptidase 20 (USP20), a DUB, doxorubicin-induced cardiotoxicity.Methods Cardiomyocyte-specific USP20-knockout (USP20-CKO) mice were utilized assess USP20 cardiomyopathy (DIC). Coimmunoprecipitation (co-IP) combined with liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) analysis employed screen substrate USP20. Furthermore, mutant plasmids constructed elucidate underlying human antigen R (HuR) by Finally, an AAV9 vector used overexpress hearts cardiac-specific HuR-knockout interaction between HuR.Results results revealed decrease expression Dox-stimulated mouse cardiomyocytes. knockout resulted increased cardiomyocyte ferroptosis led DIC. Mechanistically, directly interacted HuR through protease structural domain. Deubiquitination at position 154 crucial for maintaining stability cleaving K48 ubiquitin chains inhibiting proteasomal degradation. Additionally, bound GPX4 mRNA suppress degradation, thereby mitigating contributing alleviating targeted overexpression via cardiomyocytes alleviated However, cardiomyocyte-specific knockout, no longer had anti-DIC effect, indicating that HuR, as downstream target USP20, plays irreplaceable DIC.Conclusions Our findings indicate enhances deubiquitination,

Язык: Английский

---

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation

Cellular and Molecular Life Sciences, Год журнала: 2024, Номер 81(1)

Опубликована: Июнь 5, 2024

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and involved regulation almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with adoption a Cys-His-Asp catalytic triad. critical for recruitment endoplasmic reticulum (ER)-derived vesicles Legionella-containing vacuole (LCV). However, targets largely unknown, which restricts our understanding mechanisms used by vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins bona fide substrates SidC/SdcA. SidC/SdcA-mediated syntaxin 3 4 promotes their unconventional pairing vesicle-SNARE Sec22b, thereby contributing membrane fusion ER-derived phagosome. In addition, data reveal Rab7 its association LCV membrane. could impair binding downstream effector Rab-interacting lysosomal (RILP), partially explains why LCVs avoid lysosomes despite acquisition Rab7. Taken together, study reveals biological employed promote maturation LCVs.

Язык: Английский

---

USP9X-mediated deubiquitination of Raptor contributes to autophagy impairment and memory deficits in P301S mice

Cell Communication and Signaling, Год журнала: 2024, Номер 22(1)

Опубликована: Окт. 24, 2024

Язык: Английский

---

Preliminary exploration of the mechanisms underlying morphology engineering of Monascus purpureus during submerged fermentation via multi-omics approaches

LWT, Год журнала: 2024, Номер unknown, С. 117108 - 117108

Опубликована: Ноя. 1, 2024

Язык: Английский

---