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Highly networked signaling hubs are often associated with disease, but targeting them pharmacologically has largely been unsuccessful in the clinic because of their functional pleiotropy. Motivated by the hypothesis that a dynamic signaling code confers functional specificity, we investigated whether dynamic features may be targeted pharmacologically to achieve therapeutic specificity. With a virtual screen, we identified combinations of signaling hub topologies and dynamic signal profiles that are amenable to selective inhibition. Mathematical analysis revealed principles that may guide stimulus-specific inhibition of signaling hubs, even in the absence of detailed mathematical models. Using the NFκB signaling module as a test bed, we identified perturbations that selectively affect the response to cytokines or pathogen components. Together, our results demonstrate that the dynamics of signaling may serve as a pharmacological target, and we reveal principles that delineate the opportunities and constraints of developing stimulus-specific therapeutic agents aimed at pleiotropic signaling hubs.

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-kappaB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IkappaB, until an external stimulus triggers IkappaB degradation through an IkappaB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-kappaB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-kappaB-bound and -unbound IkappaB proteins that are critical for accurate computational predictions of steady-state IkappaB protein levels and basal NF-kappaB activity. Simulations reveal a homeostatic NF-kappaB signaling module in which differential degradation rates of free and bound pools of IkappaB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-kappaB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IkappaB, until an external stimulus triggers IkappaB degradation through an IkappaB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-kappaB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-kappaB-bound and -unbound IkappaB proteins that are critical for accurate computational predictions of steady-state IkappaB protein levels and basal NF-kappaB activity. Simulations reveal a homeostatic NF-kappaB signaling module in which differential degradation rates of free and bound pools of IkappaB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.

NF-kappa B signaling is known to be critically regulated by the NF-kappa B-inducible inhibitor protein I kappa B alpha. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-kappa B activity. We report integrated experimental and computational studies that demonstrate that another I kappa B isoform, I kappa B epsilon, also provides negative feedback on NF-kappa B activity, but with distinct functional consequences. Upon stimulation, NF-kappa B-induced transcription of I kappa B epsilon is delayed, relative to that of I kappa B alpha, rendering the two negative feedback loops to be in antiphase. As a result, I kappa B epsilon has a role in dampening I kappa B alpha-mediated oscillations during long-lasting NF-kappa B activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-kappa B activity and NF-kappa B-mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of I kappa B-NF-kappa B signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway.

NF-[kappa]B signaling is known to be critically regulated by the NF-[kappa]B-inducible inhibitor protein I[kappa]B[alpha]. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-[kappa]B activity. We report integrated experimental and computational studies that demonstrate that another I[kappa]B isoform, I[kappa]B[epsilon], also provides negative feedback on NF-[kappa]B activity, but with distinct functional consequences. Upon stimulation, NF-[kappa]B-induced transcription of I[kappa]B[epsilon] is delayed, relative to that of I[kappa]B[alpha], rendering the two negative feedback loops to be in antiphase. As a result, I[kappa]B[epsilon] has a role in dampening I[kappa]B[alpha]-mediated oscillations during long-lasting NF-[kappa]B activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-[kappa]B activity and NF-[kappa]B-mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of I[kappa]B-NF-[kappa]B signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway.

Combination of experimental and mathematical models were used to examine the complex temporal regulation phases of TNF-induced NF-[kappa]B activity, which leads to distinct gene expression programs. The results explain the nonredundant functions implied by the knockout phenotypes of i[kappa]b[alpha] and a20, and identified the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses.

This document discusses the challenges and opportunities associated with the use of biosimilars in healthcare. Biosimilars are lower-cost alternatives to biologic drugs, which are expensive and account for a significant portion of total US drug costs. The adoption of biosimilars has been hindered by factors such as lack of interchangeability with reference products, prescriber and patient misperceptions, and insurance coverage variability. The document presents a case study of a health system that developed a strategy for efficient utilization of biosimilars, resulting in decreased drug costs and enhanced reimbursement. The study highlights the importance of interdisciplinary collaboration and robust assessment of the pharmacy revenue cycle in addressing formulary management of biosimilars. [Extracted from the article]

The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for a-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction. [ABSTRACT FROM AUTHOR]

NF-κB signaling is known to be critically regulated by the NF-κB-inducible inhibitor protein IκBα. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-κB activity. We report integrated experimental and computational studies that demonstrate that another IκB isoform, IκBε, also provides negative feedback on NF-κB activity, but with distinct functional consequences. Upon stimulation, NF-κB--induced transcription of IκBε is delayed, relative to that of IκBα, rendering the two negative feedback loops to be in antiphase. As a result, IκBε has a role in dampening IκBα-mediated oscillations during long-lasting NF-κB activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-κB activity and NF-κB-mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of IκB-NF-κB signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway. [ABSTRACT FROM AUTHOR]

Inflammatory NF-kappaB/RelA activation is mediated by the three canonical inhibitors, IkappaBalpha, -beta, and -epsilon. We report here the characterization of a fourth inhibitor, nfkappab2/p100, that forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-kappaB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IkappaB protein for noncanonical NF-kappaB signaling downstream of NIK and IKK1. We develop a mathematical model of the four-IkappaB-containing NF-kappaB signaling module to account for NF-kappaB/RelA:p50 activation in response to inflammatory and developmental stimuli and find signaling crosstalk between them that determines gene-expression programs. Further combined computational and experimental studies reveal that mutant cells with altered balances between canonical and noncanonical IkappaB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.

Dynamic properties of signaling pathways control their behavior and function. We undertook an iterative computational and experimental investigation of the dynamic properties of tumor necrosis factor (TNF)alpha-mediated activation of the transcription factor NF-kappaB. Surprisingly, we found that the temporal profile of the NF-kappaB activity is invariant to the TNFalpha dose. We reverse engineered a computational model of the signaling pathway to identify mechanisms that impart this important response characteristic, thus predicting that the IKK activity profile must transiently peak at all TNFalpha doses to generate the observed NF-kappaB dynamics. Experimental confirmation of this prediction emphasizes the importance of mechanisms that rapidly down-regulate IKK following TNFalpha activation. A refined computational model further revealed signaling characteristics that ensure robust TNFalpha-mediated cell-cell communication over considerable distances, allowing for fidelity of cellular inflammatory responses in infected tissue.