The FEBS journal最新文献

The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E. coli Cascade from purified Cas proteins and analyze its activities including crRNA binding, dsDNA targeting, R-loop formation, and Cas3 recruitment. Affinity purification of 6His-tagged Cas7 coexpressed with untagged Cas5 revealed the physical association of these proteins, thus producing the Cas5-Cas7 subcomplex that was able to bind specifically to type I-E crRNA with an efficiency comparable to that of the complete Cascade. The crRNA-loaded Cas5-7 was found to bind specifically to the target dsDNA in a PAM-independent manner, albeit with a lower affinity than the complete Cascade, with both spacer sequence complementarity and repeat handles contributing to the DNA targeting specificity. The crRNA-loaded Cas5-7 targeted the complementary dsDNA with detectable formation of R-loops, which was stimulated by the addition of Cas8 and/or Cas11 acting synergistically. Cascade activity reconstitution using purified Cas5-7 and other Cas proteins showed that Cas8 was essential for specific PAM recognition, whereas the addition of Cas11 was required for Cas3 recruitment and target DNA nicking. Thus, although the core Cas5-7 subcomplex is sufficient for specific crRNA binding and basal DNA targeting, both Cas8 and Cas11 make unique contributions to efficient target recognition and cleavage.

Methyl-CpG binding protein 2 (MeCP2) is an important X-linked DNA methylation reader and a key heterochromatin organizer. The expression level of MeCP2 is crucial, as indicated by the observation that loss-of-function mutations of MECP2 cause Rett syndrome, whereas an extra copy spanning the MECP2 locus results in MECP2 duplication syndrome, both being progressive neurodevelopmental disorders. Our previous study demonstrated that MeCP2 protein expression is rapidly induced by renal ischemia-reperfusion injury (IRI) and protects the kidney from IRI through transcriptionally repressing the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 signaling pathway. However, the mechanisms underlying the upregulation of MeCP2 have remained elusive. Here, by using two hypoxia cell models, hypoxia and reoxygenation and cobalt chloride stimulation, we confirmed that the removal of lysine 48-linked ubiquitination from MeCP2 prevented its proteasome-dependent degradation under hypoxic conditions. Through unbiased screening based on a deubiquitinating enzymes library, we identified ubiquitin-specific protease 15 (USP15) as a stabilizer of MeCP2. Further studies revealed that USP15 could attenuate hypoxia-induced MeCP2 degradation by cleaving lysine 48-linked ubiquitin chains from MeCP2, primarily targeting its C-terminal domain. Consistently, USP15 inhibited hypoxia-induced signal transducer and activator of transcription 3 activation, resulting in reduced transcription of IL-6 downstream genes. In summary, our study reveals an important role for USP15 in the maintenance of MeCP2 stability and the regulation of IL-6 signaling.

Indoleamine 2,3-dioxygenase (IDO) is a monomeric heme enzyme that catalyzes the oxidative cleavage of tryptophan (L-Trp) to form N-formyl-kynurenine. Similar to other heme proteins, IDO only binds to O₂ when the heme iron is ferrous (Fe^II), thereby rendering the enzyme active. Thus, ascorbate (Asc, a reducing agent) and methylene blue (MB, an electron carrier) are commonly added to in vitro IDO assay systems. However, Asc and MB have been recently reported to significantly impact the measurement of the enzymatic parameters of vertebrate IDO. Aspergillus fumigatus is a filamentous fungus and the most common cause of invasive aspergillosis; it has three IDO genes (IDOα, IDOβ, and IDOγ). The Fe^II–O₂ IDOs of A. fumigatus, particularly Fe^II–O₂ IDOγ, have relatively long half-lives in their autoxidation; however, the autoxidation was accelerated by Asc. Similar to vertebrate IDOs, Asc acted as a competitive (or mixed-competitive) inhibitor of the IDOs of A. fumigatus. A positive correlation (in the order of IDOγ > IDOβ > IDOα) was observed between the inhibitory sensitivity of the IDOs to Asc and the facilitation of their autoxidation by Asc. The Fe^II–O₂ IDO can repeat the dioxygenase reaction as long as it reacts with L-Trp; however, substrate-free Fe^II–O₂ IDO is converted into inactive Fe^III–IDO by autoxidation. Thus, L-Trp (which keeps the IDO active) competes with Asc (which inactivates IDO by accelerating autoxidation). This is probably why Asc, which is structurally quite different from L-Trp, appears to function as a competitive (or mixed-competitive) inhibitor of IDOs.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Non-communicable diseases (NCDs), such as type 2 diabetes (T2D) and metabolic dysfunction-associated fatty liver disease, have reached epidemic proportions worldwide. The global increase in dietary sugar consumption, which is largely attributed to the production and widespread use of cheap alternatives such as high-fructose corn syrup, is a major driving factor of NCDs. Therefore, a comprehensive understanding of sugar metabolism and its impact on host health is imperative to rise to the challenge of reducing NCDs. Notably, fructose appears to exert more pronounced deleterious effects than glucose, as hepatic fructose metabolism induces de novo lipogenesis and insulin resistance through distinct mechanisms. Furthermore, recent studies have demonstrated an intricate relationship between sugar metabolism and the small intestinal microbiota (SIM). In contrast to the beneficial role of colonic microbiota in complex carbohydrate metabolism, sugar metabolism by the SIM appears to be less beneficial to the host as it can generate toxic metabolites. These fermentation products can serve as a substrate for fatty acid synthesis, imposing negative health effects on the host. Nevertheless, due to the challenging accessibility of the small intestine, our knowledge of the SIM and its involvement in sugar metabolism remains limited. This review presents an overview of the current knowledge in this field along with implications for future research, ultimately offering potential therapeutic avenues for addressing NCDs.

Bacterial toxin-antitoxin (TA) systems consist of a toxin that inhibits essential cellular processes, such as DNA replication, transcription, translation, or ATP synthesis, and an antitoxin neutralizing their cognate toxin. These systems have roles in programmed cell death, defense against phage, and the formation of persister cells. Here, we characterized the previously identified Staphylococcus aureus TA system, tsaAT, which consists of two putative membrane proteins: TsaT and TsaA. Expression of the TsaT toxin caused cell death and disrupted membrane integrity, whereas TsaA did not show any toxicity and neutralized the toxicity of TsaT. Furthermore, subcellular fractionation analysis demonstrated that both TsaA and TsaT localized to the cytoplasmic membrane of S. aureus expressing either or both 3xFLAG-tagged TsaA and 3xFLAG-tagged TsaT. Taken together, these results demonstrate that the TsaAT TA system consists of two membrane proteins, TsaA and TsaT, where TsaT disrupts membrane integrity, ultimately leading to cell death. Although sequence analyses showed that the tsaA and tsaT genes were conserved among Staphylococcus species, amino acid substitutions between TsaT orthologs highlighted the critical role of the 6th residue for its toxicity. Further amino acid substitutions indicated that the glutamic acid residue at position 63 in the TsaA antitoxin and the cluster of five lysine residues in the TsaT toxin are involved in TsaA's neutralization reaction. This study is the first to describe a bacterial TA system wherein both toxin and antitoxin are membrane proteins. These findings contribute to our understanding of S. aureus TA systems and, more generally, give new insight into highly diverse bacterial TA systems.

RETRACTION: H. Li, R. Yan, W. Chen, X. Ding, J. Liu, G. Chen, Q. Zhao, Y. Tang, S. Lv, S. Liu, and Y. Yu, “Long Non Coding RNA SLC26A4-AS1 Exerts Antiangiogenic Effects in Human Glioma by Upregulating NPTX1 Via NFKB1 Transcriptional Factor,” The FEBS Journal 288, no. 1 (2021): 212–228, https://doi.org/10.1111/febs.15325.

The above article, published online on July 15, 2020, in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief; the Federation of European Biochemical Societies; and John Wiley & Sons Ltd. The retraction has been agreed upon following an investigation into concerns raised by a third party, which revealed inappropriate image duplication between this article (Fig. 1E) and another article previously published elsewhere by a different group of authors in a different scientific context. The authors were unable to provide a satisfactory explanation, and the partial raw data they supplied could not explain the identified issues. Consequently, the editors have lost confidence in the presented data and decided to retract the paper.

Impaired kinase signalling leads to various diseases, including cancer. At the same time, kinases make up the majority of the druggable genome and targeting kinase activity has proven to be a successful first-line therapy for many cancers. Among the best-studied kinases are the mitogen-activated protein kinases (MAPKs), which regulate cell proliferation, differentiation, motility, and survival. However, the MAPK family also contains the atypical members ERK3 (MAPK6), ERK4 (MAPK4), ERK7/ERK8 (MAPK15), and NLK that are functionally and structurally different from their conventional family members and have long been neglected. Nevertheless, in recent years, important roles in carcinogenesis, actin cytoskeleton regulation and the immune system have been discovered, underlining the physiological importance of atypical MAPKs and the need to better understand their functions. This review highlights the distinctive features of the atypical MAPKs and summarizes the evidence on their regulation, physiological roles, and potential targeting strategies for cancer therapies.

Phototropin (Phot), a blue light-sensing LOV domain protein, mediates blue light responses and is evolutionarily conserved across the green lineage. Klebsormidium nitens, a green terrestrial alga, presents a valuable opportunity to study adaptive responses from aquatic to land habitat transitions. We determined the crystal structure of Klebsormidium nitens Phot LOV1 domain (KnLOV1) in the dark and engineered different mutations (R60K, Q122N, and D33N) to modulate the lifetime of the photorecovery cycle. We observed unusual, slow recovery kinetics in the wild-type KnLOV1 domain (τ = 41 ± 3 min) compared to different mutants (R60K: τ = 2.0 ± 0.1 min, Q122N: τ = 1.7 ± 0.1 min, D33N: τ = 9.6 ± 0.1 min). Crystal structures of wild-type KnLOV1 and mutants revealed subtle but critical changes near the protein chromophore that is responsible for modulating protein dark recovery time. Our findings shed light on the unique structural and biochemical characteristics of the newly studied KnLOV1 and its evolutionary importance for phototropin-mediated physiology.

Purine-pyrimidine repeats (PPRs) can form left-handed Z-form DNA and induce DNA double-strand breaks (DSBs), posing a risk for genomic rearrangements and cancer. The zinc finger (ZF) and BTB domain-containing protein 43 (ZBTB43) is a transcription factor containing two Cys2-His2 (C2H2) and one C3H1 zinc fingers and plays a crucial role in maintaining genomic and epigenomic integrity by converting mutagenic Z-form PPRs to the B-form in prospermatogonia. Despite its importance, the molecular mechanism underlying the recognition of PPRs by ZBTB43 remains elusive. In this study, we determined the X-ray crystal structure of the ZBTB43 ZF1-3 in complex with the B-form DNA containing the CA repeats sequence. The structure reveals that ZF1 and ZF2 primarily recognize the CACA sequence through specific hydrogen-bonding and van der Waals contacts via a quadruple center involving Arg389, Met411, His413, and His414. These interactions were further validated by fluorescence-based DNA-binding assays using mutated ZBTB43 variants. Our structural investigation provides valuable insights into the recognition mechanism of PPRs by ZBTB43 and suggests a potential role for ZBTB43 in the transformation of Z-DNA to B-DNA, contributing to the maintenance of genomic stability.