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Plant nucleotide binding/leucine-rich repeat (NLR) immune receptors are activated by pathogen effectors to trigger host defenses and cell death. Toll-interleukin 1 receptor domain NLRs (TNLs) converge on the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) family of lipase-like proteins for all resistance outputs. In Arabidopsis (Arabidopsis thaliana) TNL-mediated immunity, AtEDS1 heterodimers with PHYTOALEXIN DEFICIENT4 (AtPAD4) transcriptionally induced basal defenses. AtEDS1 uses the same surface to interact with PAD4-related SENESCENCE-ASSOCIATED GENE101 (AtSAG101), but the role of AtEDS1-AtSAG101 heterodimers remains unclear. We show that AtEDS1-AtSAG101 functions together with N REQUIRED GENE1 (AtNRG1) coiled-coil domain helper NLRs as a coevolved TNL cell death-signaling module. AtEDS1-AtSAG101-AtNRG1 cell death activity is transferable to the Solanaceous species Nicotiana benthamiana and cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and heterodimer structure-guided phenotyping of AtEDS1 variants and AtPAD4-AtSAG101 chimeras identify closely aligned -helical coil surfaces in the AtEDS1-AtSAG101 partner C-terminal domains that are necessary for reconstituted TNL cell death signaling. Our data suggest that TNL-triggered cell death and pathogen growth restriction are determined by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these signaling machineries coevolved with other components within plant species or clades to regulate downstream pathways in TNL immunity.

The F-box and WD repeat domain containing (FBXW) family of SCF E3 complexes has 10 members that are responsible for ubiquitination and degradation of substrate proteins involved in cell cycle regulation and tumorigenesis. Among them, FBXW1 (also called b-TrCP1/BTRC) and FBXW7 are the central proteins in this category. However, there is still a lack of elaborate exploration of the contribution of FBXW family members, especially FBXW1 and FBXW7, in various tumor types. In this present study, we preliminarily analyzed the genetic structure characteristics of the FBXW family, and systematically investigated their expression patterns and clinical correlations based on the TCGA pan-cancer data. Survival analysis of FBXWs was also conducted through the Kaplan-Meier method. In addition, we assessed their immune infiltration level through immune-related algorithms like Timer and xCell. There were obvious genetic heterogeneity and different clinical traits in FBXW family members. Moreover, we found that FBXW family genes may be useful in predicting prognosis and therapeutic efficacy using survival analysis. In addition, the immune infiltration of FBXW family was also clearly illustrated in this study. The results showed these genes were closely involved in immune components such as immune score, immune subtypes, tumor-infiltrating lymphocytes and immune checkpoints. Notedly, FBXW1 as an oncogene and FBXW7 as a tumor suppressor gene also show opposite relationships on immune cells. Our results provided valuable strategies to guide the therapeutic orientation concerning the role of FBXW family genes in cancer.

Trace elements play important roles in human health, but little is known about their functions in humoral immunity. Here, we show an important role for iron in inducing cyclin E and B cell proliferation. We find that iron-deficient individuals exhibit a significantly reduced antibody response to the measles vaccine when compared to iron-normal controls. Mice with iron deficiency also exhibit attenuated T-dependent or T-independent antigen-specific antibody responses. We show that iron is essential for B cell proliferation; both iron deficiency and α-ketoglutarate inhibition could suppress cyclin E1 induction and S phase entry of B cells upon activation. Finally, we demonstrate that three demethylases, KDM2B, KDM3B and KDM4C, are responsible for histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter, cyclin E1 induction and B cell proliferation. Thus, our data reveal a crucial role of H3K9 demethylation in B cell proliferation, and the importance of iron in humoral immunity. The authors show an important role for iron in B cell proliferation via histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter. Using a measles vaccination murine model, they show that iron-deficient individuals have a significantly reduced antibody response to the vaccine when compared to iron-normal controls.

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE–expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein. Significance The identification of specific ubiquitin ligase–substrate pairs is crucial for understanding the roles of protein ubiquitination in the regulation of diverse biological processes. Despite the development of various methodologies for substrate identification, it remains challenging to determine ubiquitin ligase substrates. Based on previously described tandem ubiquitin-binding entity(ies) (TUBE), we designed the trypsin-resistant (TR)-TUBE for expression in cells. The coexpression of TR-TUBE with an ubiquitin ligase stabilizes the ubiquitinated substrates by masking the ubiquitin chains. Using a combination of two strategies for enriching ubiquitinated substrates, TR-TUBE and anti–Lys-ε-Gly-Gly antibody, we successfully identified specific ubiquitin ligase–substrate pairs. Our methodology provides an effective means for the identification of ubiquitin ligase substrates and the detection of ubiquitin ligase activity.

As critical substrate-recognition subunits of the SCF (SKP1-CUL1-F-box) ubiquitin ligase complex, F-box proteins mediate the ubiquitination and subsequent degradation of specific target proteins, playing pivotal roles in cell cycle regulation, signal transduction (e.g., MAPK and NF-κB pathways), and immune homeostasis. F-box proteins have dual regulatory functions in tumorigenesis and immune escape. On one hand, their expression is dynamically modulated by upstream signaling pathways (including PI3K/AKT and Wnt/β-catenin cascades) and epigenetic modifications (such as DNA methylation and histone acetylation), thereby influencing the stability of oncogenic factors (e.g., c-MYC, Cyclin E) or tumor suppressors (e.g., p53). On the other hand, F-box proteins directly regulate tumor immune microenvironments by targeting immune-related molecules for degradation, thereby modulating T-cell activation, macrophage polarization, and immune checkpoint functionality (specifically PD-1/PD-L1 axis and CTLA-4 signaling). This review systematically summarizes the upstream and downstream regulatory networks of F-box proteins, with an emphasis on their molecular mechanisms in tumor immunosuppression. It highlights the potential strategies and drug resistance mechanisms in targeting F-box proteins for combination with immunotherapies, while also discussing future research applications and development directions of F-box proteins. These insights aim to advance the development of novel immunotherapeutic strategies for precision cancer treatment.

Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity. However, key regulators of ubiquitination during innate response and roles of new types of ubiquitination (apart from Lys48- and Lys63-linkage) in control of innate signaling have not been clearly understood. Here we report that F-box only protein Fbxo21, a functionally unknown component of SCF (Skp1–Cul1–F-box protein) complex, facilitates Lys29-linkage and activation of ASK1 (apoptosis signal-regulating kinase 1), and promotes type I interferon production upon viral infection. Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1, attenuates c-Jun N-terminal kinase (JNK) and p38 signaling pathway, and decreases the production of proinflammatory cytokines and type I interferon, resulting in reduced antiviral innate response and enhanced virus replication. Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response, providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response. The innate immune system is the body’s first line of defense against being infected by viruses and other microbes. Upon recognizing a virus, host cells trigger the innate immune response in an effort to eliminate the threat. However, innate immune responses must be carefully controlled because an excessive response can cause inflammation that harms the body. The innate immune response involves a variety of cells and processes that are each activated through a series of communication systems called signaling pathways. While much has been learned about which parts of a virus trigger the innate immune response, it is not clear how the immune response to the virus is controlled. It has been suggested that a process known as ubiquitination could be involved in regulating the activity of signaling pathways that activate the innate immune response. During ubiquitination, enzymes attach a small molecule called ubiquitin to a specific target protein. Ubiquitin often acts as a label that targets a particular protein for destruction. Enzymes called E3 ubiquitin ligases play central roles in identifying specific target proteins for ubiquitination. Some of these enzymes consist of a single protein unit that acts alone, but other E3 ubiquitin ligases are formed by groups (or “complexes”) of several proteins working together. Members of the F-box only protein family are components of some ubiquitin ligase complexes. Here, Yu et al. used a “microarray” technique to assess which F-box only proteins in mice are produced during an immune response to two viruses. The experiments identified an F-box protein called Fbxo21 as a potential candidate for a role in regulating the innate immune response. Additional experiments revealed that Fbxo21 is involved in adding ubiquitin to a specific location on a signaling protein called ASK1, which is known to be crucial for innate immune responses. Instead of targeting ASK1 for destruction, this ubiquitination activates ASK1. Therefore, Yu et al.’s findings demonstrate that Fbxo21 plays an important role in regulating innate immune responses. A future challenge is to investigate exactly how ASK1 is activated by the ubiquitin.

Little is known about the regulation of the ST2L receptor for IL-33. Zhao and colleagues identify the F-box protein FBXL19 as being key to the degradation and negative regulation of ST2L. The ST2L receptor for interleukin 33 (IL-33) mediates pulmonary inflammation and immune system–related disorders, such as asthma and rheumatoid arthritis. At present, very little is known about the molecular regulation of ST2L expression. Here we found that FBXL19, an 'orphan' member of the Skp1–Cullin–F-box family of E3 ubiquitin ligases, selectively bound to ST2L to mediate its polyubiquitination and elimination in the proteasome. Degradation of ST2L involved phosphorylation of ST2L at Ser442 catalyzed by the kinase GSK3β. Overexpression of FBXL19 abrogated the proapoptotic and inflammatory effects of IL-33 and lessened the severity of lung injury in mouse models of pneumonia. Our results suggest that modulation of the IL-33–ST2L axis by ubiquitin ligases might serve as a unique strategy for lessening pulmonary inflammation.

Post-acute sequelae of SARS-CoV-2 infection (PASC), commonly known as “Long COVID”, represents a significant clinical challenge characterized by persistent symptoms following acute COVID-19 infection. We conducted a comprehensive retrospective cohort study to identify serum autoantibody biomarkers associated with PASC. Initial screening using a protein bead array comprising approximately 20,000 human proteins identified several candidate PASC-associated autoantibodies. Subsequent validation by enzyme-linked immunosorbent assay (ELISA) in an expanded cohort—consisting of PASC patients, non-PASC COVID-19 convalescents, and pre-pandemic healthy controls—revealed two promising biomarkers: autoantibodies targeting PITX2 and FBXO2. PITX2 autoantibodies demonstrated high accuracy in distinguishing PASC patients from both non-PASC convalescents (area under the curve [AUC] = 0.891) and healthy controls (AUC = 0.866), while FBXO2 autoantibodies showed moderate accuracy (AUC = 0.762 and 0.786, respectively). Notably, the levels of these autoantibodies were associated with several PASC symptoms, including fever, dyspnea, palpitations, loss of appetite, and brain fog. The identification of PITX2 and FBXO2 autoantibodies as biomarkers not only enhances our understanding of PASC pathophysiology but also provides promising candidates for further investigation.

Bacterial pathogens often manipulate host immune pathways to establish acute and chronic infection. Many Gram-negative bacteria do this by secreting effector proteins through a type III secretion system that alter the host response to the pathogen. In this study, we determined that the phage-encoded GogB effector protein in Salmonella targets the host SCF E3 type ubiquitin ligase through an interaction with Skp1 and the human F-box only 22 (FBXO22) protein. Domain mapping and functional knockdown studies indicated that GogB-containing bacteria inhibited IκB degradation and NFκB activation in macrophages, which required Skp1 and a eukaryotic-like F-box motif in the C-terminal domain of GogB. GogB-deficient Salmonella were unable to limit NFκB activation, which lead to increased proinflammatory responses in infected mice accompanied by extensive tissue damage and enhanced colonization in the gut during long-term chronic infections. We conclude that GogB is an anti-inflammatory effector that helps regulate inflammation-enhanced colonization by limiting tissue damage during infection.

Cryptococcus neoformans is the main etiologic agent of cryptococcal meningitis and causes a significant number of deadly infections per year. Although it is well appreciated that host immune responses are crucial for defense against cryptococcosis, our understanding of factors that control the development of effective immunity to this fungus remains incomplete. In previous studies, we identified the F-box protein Fbp1 as a novel determinant of C. neoformans virulence. In this study, we found that the hypovirulence of the fbp1 Δ mutant is linked to the development of a robust host immune response. Infection with the fbp1 Δ mutant induces a rapid influx of CCR2 + monocytes and their differentiation into monocyte-derived dendritic cells (mo-DCs). Depletion of CCR2 + monocytes and their derivative mo-DCs resulted in impaired activation of a protective inflammatory response and the rapid death of mice infected with the fbp1 Δ mutant. Mice lacking B and T cells also developed fungal meningitis and succumbed to infection with the fbp1 Δ mutant, demonstrating that adaptive immune responses to the fbp1 Δ mutant help to maintain the long-term survival of the host. Adaptive immune responses to the fbp1 Δ mutant were characterized by enhanced differentiation of Th1 and Th17 CD4 + T cells together with diminished Th2 responses compared to the H99 parental strain. Importantly, we found that the enhanced immunogenicity of fbp1 Δ mutant yeast cells can be harnessed to confer protection against a subsequent infection with the virulent H99 parental strain. Altogether, our findings suggest that Fbp1 functions as a novel virulence factor that shapes the immunogenicity of C. neoformans . IMPORTANCE Cryptococcus neoformans is the most common cause of deadly fungal meningitis, with over 270,000 infections per year. Immune responses are critically required for the prevention of cryptococcosis, and patients with impaired immunity and low CD4 + T cell numbers are at high risk of developing these deadly infections. Although it is well appreciated that the development of protective immunity is shaped by the interactions of the host immune system with fungal cells, our understanding of fungal products that influence this process remains poor. In this study, we found that the activity of F-box protein 1 (Fbp1) in highly virulent C. neoformans clinical strain H99 shapes its immunogenicity and thus affects the development of protective immune responses in the host. The identification of this new mechanism of virulence may facilitate the future development of therapeutic interventions aimed at boosting antifungal host immunity. Cryptococcus neoformans is the most common cause of deadly fungal meningitis, with over 270,000 infections per year. Immune responses are critically required for the prevention of cryptococcosis, and patients with impaired immunity and low CD4 + T cell numbers are at high risk of developing these deadly infections. Although it is well appreciated that the development of protective immunity is shaped by the interactions of the host immune system with fungal cells, our understanding of fungal products that influence this process remains poor. In this study, we found that the activity of F-box protein 1 (Fbp1) in highly virulent C. neoformans clinical strain H99 shapes its immunogenicity and thus affects the development of protective immune responses in the host. The identification of this new mechanism of virulence may facilitate the future development of therapeutic interventions aimed at boosting antifungal host immunity.