The aryl hydrocarbon receptor and FOS mediate cytotoxicity induced by Acinetobacter baumannii.

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.

Chem-CRISPR/dCas9FCPF: a platform for chemically induced epigenome editing.

Epigenetic aberration is one of the major driving factors in human cancer, often leading to acquired resistance to chemotherapies. Various small molecule epigenetic modulators have been reported. Nonetheless, outcomes from animal models and clinical trials have underscored the substantial setbacks attributed to pronounced on- and off-target toxicities. To address these challenges, CRISPR/dCas9 technology is emerging as a potent tool for precise modulation of epigenetic mechanism. However, this technology involves co-expressing exogenous epigenetic modulator proteins, which presents technical challenges in preparation and delivery with potential undesirable side effects. Recently, our research demonstrated that Cas9 tagged with the Phe-Cys-Pro-Phe (FCPF)-peptide motif can be specifically targeted by perfluorobiphenyl (PFB) derivatives. Here, we integrated the FCPF-tag into dCas9 and established a chemically inducible platform for epigenome editing, called Chem-CRISPR/dCas9FCPF. We designed a series of chemical inhibitor-PFB conjugates targeting various epigenetic modulator proteins. Focusing on JQ1, a panBET inhibitor, we demonstrate that c-MYC-sgRNA-guided JQ1-PFB specifically inhibits BRD4 in close proximity to the c-MYC promoter/enhancer, thereby effectively repressing the intricate transcription networks orchestrated by c-MYC as compared with JQ1 alone. In conclusion, our Chem-CRISPR/dCas9FCPF platform significantly increased target specificity of chemical epigenetic inhibitors, offering a viable alternative to conventional fusion protein systems for epigenome editing.

Membrane remodeling via ubiquitin-mediated pathways.

The dynamic process of membrane shaping and remodeling plays a vital role in cellular functions, with proteins and cellular membranes interacting intricately to adapt to various cellular needs and environmental cues. Ubiquitination-a posttranslational modification-was shown to be essential in regulating membrane structure and shape. It influences virtually all pathways relying on cellular membranes, such as endocytosis and autophagy by directing protein degradation, sorting, and oligomerization. Ubiquitin is mostly known as a protein modifier; however, it was reported that ubiquitin and ubiquitin-like proteins can associate directly with lipids, affecting membrane curvature and dynamics. In this review, we summarize some of the current knowledge on ubiquitin-mediated membrane remodeling in the context of endocytosis, autophagy, and ER-phagy.

Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis.

The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner.Abbreviation: ARE: antioxidant response element; Dup: deubiquitinase specific for phosphoribosyl-linked serine ubiquitination; ER: endoplasmic reticulum; h.p.i: hours post infection; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; KEAP1: kelch like ECH associated protein 1; KIR: KEAP1-interacting region; LIR: LC3-interacting region; NES: nuclear export signal; NFKB/NF-κB: nuclear factor kappa B; NLS: nuclear localization signal; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1 domain: Phox1 and Bem1 domain; PR-Ub: phosphoribosyl-linked serine ubiquitination; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; tBHQ: tertiary butylhydroquinone; TUBE2: tandem ubiquitiin binding entity 2; UBA domain: ubiquitin-associated domain.

Covalent Probes To Capture Legionella pneumophila Dup Effector Enzymes.

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires' disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and X-ray crystallography approaches were used to identify the site of covalent cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

C16ORF70/MYTHO promotes healthy aging in C.elegans and prevents cellular senescence in mammals.

The identification of genes that confer either extension of life span or accelerate age-related decline was a step forward in understanding the mechanisms of aging and revealed that it is partially controlled by genetics and transcriptional programs. Here, we discovered that the human DNA sequence C16ORF70 encodes a protein, named MYTHO (macroautophagy and youth optimizer), which controls life span and health span. MYTHO protein is conserved from Caenorhabditis elegans to humans and its mRNA was upregulated in aged mice and elderly people. Deletion of the orthologous myt-1 gene in C. elegans dramatically shortened life span and decreased animal survival upon exposure to oxidative stress. Mechanistically, MYTHO is required for autophagy likely because it acts as a scaffold that binds WIPI2 and BCAS3 to recruit and assemble the conjugation system at the phagophore, the nascent autophagosome. We conclude that MYTHO is a transcriptionally regulated initiator of autophagy that is central in promoting stress resistance and healthy aging.

BAG3 regulates cilia homeostasis of glioblastoma via its WW domain.

The multidomain protein BAG3 exerts pleiotropic oncogenic functions in many tumor entities including glioblastoma (GBM). Here, we compared BAG3 protein-protein interactions in either adherently cultured or stem-like cultured U251 GBM cells. In line with BAG3's putative role in regulating stem-like properties, identified interactors in sphere-cultured cells included different stem cell markers (SOX2, OLIG2, and NES), while interactomes of adherent BAG3-proficient cells indicated a shift toward involvement of BAG3 in regulation of cilium assembly (ACTR3 and ARL3). Applying a set of BAG3 deletion constructs we could demonstrate that none of the domains except the WW domain are required for suppression of cilia formation by full-length BAG3 in U251 and U343 cells. In line with the established regulation of the Hippo pathway by this domain, we could show that the WW mutant fails to rescue YAP1 nuclear translocation. BAG3 depletion reduced activation of a YAP1/AURKA signaling pathway and induction of PLK1. Collectively, our findings point to a complex interaction network of BAG3 with several pathways regulating cilia homeostasis, involving processes related to ciliogenesis and cilium degradation.

Correction: Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.

[This corrects the article DOI: 10.1371/journal.pone.0253364.].

Targeted protein degradation via intramolecular bivalent glues.

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

TBC1D2B undergoes phase separation and mediates autophagy initiation.

Small ubiquitin-like modifiers from the ATG8 family regulate autophagy initiation and progression in mammalian cells. Their interaction with LC3-interacting region (LIR) containing proteins promotes cargo sequestration, phagophore assembly, or even fusion between autophagosomes and lysosomes. Previously, we have shown that RabGAP proteins from the TBC family directly bind to LC3/GABARAP proteins. In the present study, we focus on the function of TBC1D2B. We show that TBC1D2B contains a functional canonical LIR motif and acts at an early stage of autophagy by binding to both LC3/GABARAP and ATG12 conjugation complexes. Subsequently, TBC1D2B is degraded by autophagy. TBC1D2B condensates into liquid droplets upon autophagy induction. Our study suggests that phase separation is an underlying mechanism of TBC1D2B-dependent autophagy induction.

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.

Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails ("Ala-tails") that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 RING ligase2)-KLHDC10. Here, we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails and that structural predictions identify candidate Ala-tail-binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail-binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron-recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.

Ubiquitination and cell-autonomous immunity.

Cell-autonomous immunity is the first line of defense by which cells recognize and contribute to eliminating invasive pathogens. It is composed of immune signaling networks that sense microbial pathogens, promote pathogen restriction, and stimulate their elimination, including host cell death. Ubiquitination is a pivotal orchestrator of these pathways, by changing the activity of signal transducers and effector proteins in an efficient way. In this review, we will focus on how ubiquitin connects the pathways that sense pathogens to the cellular responses to invaders and shed light on how ubiquitination impacts the microenvironment around the infected cell, stimulating the appropriate immune response. Finally, we discuss therapeutic options directed at favoring cell-autonomous immune responses to infection.

A Pro-Fluorescent Ubiquitin-Based Probe to Monitor Cysteine-Based E3 Ligase Activity.

Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.

Pathogenetic mechanisms and therapeutic approaches of acute-to-chronic liver failure.

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

The mission to ensure continued funding for excellent basic research.

The surprising decision by Novo Nordisk Foundation (NNF) to discontinue funding for the Center for Protein Research in Copenhagen should prompt discussions about public and private commitment to support basic research.