EMBO Reports最新文献

The SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex plays a protective role against external stress, such as ultraviolet irradiation. The emergence of substrates activates SCF through neddylation, the covalent attachment of ubiquitin-like protein NEDD8 to CUL1. After substrate degradation, SCF is inactivated through deneddylation by COP9-signalosome (CSN), a solo enzyme that can deneddylate SCF. How the activity of CSN and SCF is coordinated within the cell is not fully understood. Here, we find that heat-shock cognate 70 (HSC70) chaperone coordinates SCF and CSN activation dependent on the neddylation status and substrate availability. Under basal conditions and low substrate availability, HCS70 directly enhances CSN deneddylation activity, thereby reducing SCF activity. Under SCF-activated conditions, HSC70 interacts with neddylated SCF and enhances its ubiquitination activity. The alternative interaction between HSC70 and CSN or neddylated SCF is regulated by the presence or absence of SCF substrates. The knockdown of HSC70 decreases SCF-mediated substrate ubiquitination, resulting in vulnerability against ultraviolet irradiation. Our work demonstrates the pivotal role of HSC70 in the alternative activation of CSN deneddylation and SCF substrate ubiquitination, which enables a prompt stress response.

Immune responses play an integral role in skeletal muscle regeneration. In the genetically inherited muscle disease Duchenne muscular dystrophy (DMD), muscle regeneration is disrupted, leading to chronic inflammation, fibrosis, and early mortality. Previously, it has been suggested that type-2 innate immune cells, particularly eosinophils and their production of IL-4, play an essential role in effective muscle regeneration after acute injury. We here re-investigate the role of eosinophils in skeletal muscle repair using mice deficient in eosinophils (ΔdblGATA), or deficient in IL-4R/IL-13R signaling through STAT6 (Stat6-/-). We show that neither deficiency has an impact on skeletal muscle regeneration in response to acute injury as quantified by fiber size, immune cell infiltration, or muscle-resident stem cell proliferation. We also investigate the role of STAT6 signaling in mdx:Stat6-/- mice, a model of DMD and, again, find that ablation of STAT6 signaling has no effect on the rate or severity of fibrotic scar formation or disease progression. In contrast to previous models, our data suggest a negligible role for eosinophils and STAT6 signaling in skeletal muscle regeneration after acute or chronic injury.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNALP) is essential for the immortalization of naive B lymphocytes (NBLs). However, the mechanisms remain elusive. To understand EBNALP's role in B-cell transformation, we compare NBLs infected with wild-type EBV and an EBNALP-null mutant EBV using multi-omics techniques. EBNALP inactivation alters enhancer-promoter interactions, resulting in decreased CCND2 and increased CASP1 and BCL2L11 expression. Mechanistically, EBNALP interacts with and colocalizes with the looping factor YY1. Depletion of EBNALP reduces YY1 DNA-binding and enhancer-promoter interactions, similar to effects observed with YY1 depletion. Furthermore, EBNALP colocalizes with DPF2, a protein that binds to H3K14ac and H4K16ac. CRISPR depletion of DPF2 reduces both EBNALP and YY1 DNA binding, suggesting that the DPF2/EBNALP complex may tether YY1 to DNA to increase enhancer-promoter interactions. EBNALP inactivation also increases enhancer-promoter interactions at the CASP1 and BCL2L11 loci, along with elevated DPF2 and YY1 binding and DNA accessibility. Our data suggest that EBNALP regulates YY1 to rewire the host genome, which might facilitate naive B-cell transformation.

Thermogenic fat, including brown and beige fat, dissipates heat via thermogenesis and enhances energy expenditure. Thus, its activation represents a therapeutic strategy to combat obesity. Here, we demonstrate that levels of F-box and WD repeat domain-containing 7 (FBXW7), an E3 ubiquitin protein ligase, negatively correlate with thermogenic fat functionality. FBXW7 overexpression in fat suppresses energy expenditure and thermogenesis, thus aggravates obesity and metabolic dysfunctions in mice. Conversely, FBXW7 depletion in fat leads to brown fat expansion and browning of white fat, and protects mice from diet induced obesity, hepatic steatosis, and hyperlipidemia. Mechanistically, FBXW7 binds to S6K1 and promotes its ubiquitination and proteasomal degradation, which in turn impacts glycolysis and brown preadipocyte proliferation via lactate. Besides, the beneficial metabolic effects of FBXW7 depletion in fat are attenuated by fat-specific knockdown of S6K1 in vivo. In summary, we provide evidence that adipose FBXW7 acts as a major regulator for thermogenic fat biology and energy homeostasis and serves as potential therapeutic target for obesity and metabolic diseases.

Histone deacetylase HDAC6 has been implicated in regulating antiviral innate immunity. However, its precise function in response to DNA virus infection remains elusive. Herein, we find that HDAC6 deficiency promotes the activation of cGAS-STING signaling and type I interferon (IFN) production, both in vitro and in vivo, resulting in a decrease in HSV-1 infection. Mechanistically, HDAC6 deacetylates tripartite motif protein 56 (TRIM56) at K110 in mice, thereby impairing the monoubiquitination cGAS and its DNA binding ability. Overexpression of TRIM56 K110Q protects mice against HSV-1 infection. Notably, different amino acids at position 110 of TRIM56 in human and mouse cause species-specific IFN responses. Further, we show that during early stages of HSV-1 infection, the viral protein US3 phosphorylates HDAC6 to inhibit the cGAS-mediated antiviral response. Our results suggest that HDAC6 inhibits cGAS activation through TRIM56 deacetylation in a species-specific manner.

Spindle assembly checkpoint (SAC) inhibitors are a recently developed class of drugs, which perturb chromosome segregation during cell division, induce chromosomal instability (CIN), and eventually lead to cell death. The molecular features that determine cellular sensitivity to these drugs are not fully understood. We recently reported that aneuploid cancer cells are preferentially sensitive to SAC inhibition. Here we report that sensitivity to SAC inhibition by MPS1 inhibitors is largely driven by the expression of CDC20, a main mitotic activator of the anaphase-promoting complex (APC/C), and that the effect of CDC20 is larger than that of the APC/C itself. Mechanistically, we discovered that CDC20 depletion prolongs metaphase duration, diminishes mitotic errors, and reduces sensitivity to SAC inhibition. We found that aneuploid cells express higher basal levels of CDC20, which shortens the duration of metaphase and leads to multiple mitotic errors, resulting in increased long-term sensitivity to the additional CIN induced by SAC inhibition. Our findings propose high CDC20 expression as a molecular feature associated with the sensitivity to SAC inhibition therapy and as a potential aneuploidy-induced cellular vulnerability.

Salmonella enterica serotype Typhimurium (Salmonella) resides and multiplies intracellularly in cholesterol-rich compartments called Salmonella-containing vacuoles (SCVs) with actin-rich tubular extensions known as Salmonella-induced filaments (SIFs). SCV maturation depends on host-derived cholesterol, but the transport mechanism of low-density lipoprotein (LDL)-derived cholesterol to SCVs remains unclear. Here we find that peroxisomes are recruited to SCVs and function as pro-bacterial organelle. The Salmonella effector protein SseI is required for the interaction between peroxisomes and the SCV. SseI contains a variant of the PTS1 peroxisome-targeting sequence, GKM, localizes to the peroxisomes and activates the host Ras GTPase, ADP-ribosylation factor-1 (ARF-1). Activation of ARF-1 leads to the recruitment of phosphatidylinsolitol-5-phosphate-4 kinase and the generation of phosphatidylinsolitol-4-5-bisphosphate on peroxisomes. This enhances the interaction of peroxisomes with lysosomes and allows for the transfer of lysosomal cholesterol to SCVs using peroxisomes as a bridge. Salmonella infection of peroxisome-depleted cells leads to the depletion of cholesterol on the SCVs, resulting in reduced SIF formation and bacterial proliferation. Taken together, our work identified peroxisomes as a target of Salmonella secretory effectors, and as conveyance of host cholesterol to enhance SCV stability, SIF integrity, and intracellular bacterial growth.

Import and assembly of mitochondrial proteins into multimeric complexes are essential for cellular function. Yet, many steps of these processes and the proteins involved remain unknown. Here, we identify a novel pathway for disulfide bond formation and assembly of mitochondrial inner membrane (IM) proteins. Dbi1, a previously uncharacterized IM protein, interacts with an unassembled pool of Tim17, the central subunit of the presequence translocase of the IM, and is upregulated in cells with increased levels of unassembled Tim17. In the absence of Dbi1, the conformation of the presequence translocase is affected and stability of Tim17 is reduced. Furthermore, Dbi1, through its conserved CxxC motif, is involved in the formation of the disulfide bond in Tim17 in a manner independent of the disulfide relay system, the major oxidation-driven protein import pathway into mitochondria. The substrate spectrum of Dbi1 is not limited to Tim17 but includes at least two more IM proteins, Tim22 and Cox20. We conclude that Dbi1 is a novel oxidoreductase in mitochondria which introduces disulfide bonds into IM proteins and chaperones their assembly into multimeric protein complexes.

Neuroligins are postsynaptic cell-adhesion molecules that regulate synaptic function with a remarkable isoform specificity. Although Nlgn1 and Nlgn2 are highly homologous and biochemically interact with the same extra- and intracellular proteins, Nlgn1 selectively functions in excitatory synapses whereas Nlgn2 functions in inhibitory synapses. How this excitatory/inhibitory (E/I) specificity arises is unknown. Using a comprehensive structure-function analysis, we here expressed wild-type and mutant neuroligins in functional rescue experiments in cultured hippocampal neurons lacking all endogenous neuroligins. Electrophysiology confirmed that Nlgn1 and Nlgn2 selectively restored excitatory and inhibitory synaptic transmission, respectively, in neuroligin-deficient neurons, aligned with their synaptic localizations. Chimeric Nlgn1-Nlgn2 constructs reveal that the extracellular neuroligin domains confer synapse specificity, whereas their intracellular sequences are exchangeable. However, the cytoplasmic sequences of Nlgn2, including its Gephyrin-binding motif that is identically present in the Nlgn1, is essential for its synaptic function whereas they are dispensable for Nlgn1. These results demonstrate that although the excitatory vs. inhibitory synapse specificity of Nlgn1 and Nlgn2 are both determined by their extracellular sequences, these neuroligins enable normal synaptic connections via distinct intracellular mechanisms.