The NF-κB signaling system plays an important regulatory role in the control of many biological processes. The activities of NF-κB signaling networks and the expression of their target genes are frequently elevated in pathophysiological situations including inflammation, infection, and cancer. In these conditions, the outcome of NF-κB activity can vary according to (i) differential activation states, (ii) the pattern of genomic recruitment of the NF-κB subunits, and (iii) cellular heterogeneity. Additionally, the cytosolic NF-κB activation steps leading to the liberation of DNA-binding dimers need to be distinguished from the less understood nuclear pathways that are ultimately responsible for NF-κB target gene specificity. This raises the need to more precisely determine the NF-κB activation status not only for the purpose of basic research, but also in (future) clinical applications. Here we review a compendium of different methods that have been developed to assess the NF-κB activation status in vitro and in vivo. We also discuss recent advances that allow the assessment of several NF-κB features simultaneously at the single cell level.

As cancer cell specific rewiring of metabolic networks creates potential therapeutic opportunities, we conducted a synthetic lethal screen utilizing inhibitors of metabolic pathways. Simultaneous administration of (R)-GNE-140 and BMS-986205 (Linrodostat) preferentially halted proliferation of ovarian cancer cells, but not of their non-oncogenically transformed progenitor cells. While (R)-GNE-140 inhibits lactate dehydrogenase (LDH)A/B and thus effective glycolysis, BMS-986205, in addition to its known inhibitory activity on Indoleamine 2,3-dioxygenase (IDO1), also restricts oxidative phosphorylation (OXPHOS), as revealed here. BMS-986205, which is being tested in multiple Phase III clinical trials, inhibits the ubiquinone reduction site of respiratory complex I and thus compromises mitochondrial ATP production. The energetic catastrophe caused by simultaneous interference with glycolysis and OXPHOS resulted in either cell death or the induction of senescence in tumor cells, with the latter being eliminated by senolytics. The frequent synergy observed with combined inhibitor treatment was comprehensively confirmed through testing on tumor cell lines from the DepMap panel and on human colorectal cancer organoids. These experiments revealed highly synergistic activity of the compounds in a third of the tested tumor cell lines, correlating with alterations in genes with known roles in metabolic regulation and demonstrating the therapeutic potential of metabolic intervention.

The NF-κB system is a key transcriptional pathway that regulates innate and adaptive immunity because it triggers the activation and differentiation processes of lymphocytes and myeloid cells during immune responses. In most instances, binding to cytoplasmic inhibitory IκB proteins sequesters NF-κB into an inactive state, while a plethora of external triggers activate three complex signaling cascades that mediate the release and nuclear translocation of the NF-κB DNA-binding subunits. In addition to these cytosolic steps (level 1 of NF-κB regulation), NF-κB activity is also controlled in the nucleus by signaling events, cofactors and the chromatin environment to precisely determine chromatin recruitment and the specificity and timing of target gene transcription (level 2 of NF-κB regulation). Here, we discuss an additional layer of the NF-κB system that manifests in various steps of post-transcriptional gene expression and protein secretion. This less-studied regulatory level allows reduction of (transcriptional) noise and signal integration and endows time-shifted control of the secretion of inflammatory mediators. Detailed knowledge of these steps is important, as dysregulated post-transcriptional NF-κB signaling circuits are likely to foster chronic inflammation and contribute to the formation and maintenance of a tumor-promoting microenvironment.

The functionally redundant ubiquitin E3 ligases SIAH1 and SIAH2 have been implicated in the regulation of metabolism and the hypoxic response, while their role in other signal-mediated processes such inflammatory gene expression remains to be defined. Here we have downregulated the expression of both SIAH proteins with specific siRNAs and investigated the functional consequences for IL-1α-induced gene expression. The knockdown of SIAH1/2 modulated the expression of approximately one third of IL-1α-regulated genes. These effects were not due to changes in the NF-κB and MAPK signaling pathways and rather employed further processes including those mediated by the coactivator p300. Most of the proteins encoded by SIAH1/2-regulated genes form a regulatory network of proinflammatory factors. Thus SIAH1/2 proteins function as variable rheostats that control the amplitude rather than the principal activation of the inflammatory gene response.

Background: The kernel number of maize (Zea mays L.) at maturity is mainly determined at the time around pollination. Kernel abortion frequently occurs during this period, leading to grain yield depressions. Plasma membrane (PM) H⁺‐ATPase was identified as a key enzyme responsible for supply of assimilates to the developing maize kernels shortly after pollination.Aims: This study aimed at stimulating PM H⁺‐ATPase activity in the kernels by in vivo application of the auxin indole‐3‐acetic acid (IAA) to maize plants at flowering, leading to an improved hexose uptake and finally to a better kernel set.Methods: Maize plants were cultivated under well‐watered conditions using the container technique. IAA was applied to unstressed maize plants twice, 2 days before controlled pollination and at pollination (application rate per plant: 1.9 mL of 1.5 mM IAA). The developing kernels were harvested 2 days after pollination, and PM vesicles were isolated and purified using two‐phase partitioning.Results: The in vitro hydrolytic activity of the PM H⁺‐ATPase was significantly stimulated by 22% due to in vivo IAA application (control: 0.99 ± 0.05, IAA treatment: 1.21 ± 0.03* μmol inorganic phosphate mg^–1 protein min^–1). V_max was significantly increased by IAA treatment, whereas K_m was reduced. The maximal pH gradient (ΔA₄₉₂) at the PM was increased by 10% (control: 0.071 ± 0.002, IAA treatment: 0.078 ± 0.002*). IAA caused a significant increase of PM H⁺‐ATPase abundance in the vesicles. Concentrations of sucrose and hexoses as well as acid invertase activity in the kernels were unaffected by IAA treatment. However, at maturity kernel numbers per cob were significantly decreased causing grain yield reductions of 19%.Conclusions: Increased PM H⁺‐ATPase activity could not be translated into grain yield improvements. Probably the auxin application occurred too early during kernel development. As cytokinins play a key role during pollination, auxin application at this stage may have disturbed the phytohormone balance, causing disruption of cell division and a rather early onset of cell extension due to increased IAA concentrations. In further studies, it should be tested if applications of cytokinin at flowering or of IAA at a later growth stage have positive impacts on kernel set.

The well-studied NF-κB signaling system is a key mediator of the inflammatory response. Large-scale sequencing studies in humans now allow initial insights into non-essential human genes in which both alleles carry mutations that prevent protein expression or function. Here, we compiled the non-essential genes identified in various sequencing studies and analyzed the occurrence of knockouts in the human NF-κB signaling system. This revealed a lower knockout frequency in the NF-κB system compared to the entire genome. Since drugs inhibiting NF-κB pathway components were unsuccessful in clinical trials so far, the naturally occurring knockouts of NF-κB and its upstream regulators could provide new candidates for therapeutic intervention. To investigate the potential functional importance of posttranslational modifications (PTMs) occurring on NF-κB components, we analyzed not only their evolutionary conservation but also, as a second criterion, their genetic constraint in the sequenced individuals. This approach revealed the absence of missense mutations at key modification sites involved in NF-κB activation and identified additional candidate sites for future studies.