Seth A. Brooks

The Role of mRNA Turnover in the Regulation of Tristetraprolin Expression: Evidence for an Extracellular Signal-Regulated Kinase-Specific, AU-Rich Element-Dependent, Autoregulatory Pathway

Tristetraprolin (TTP) is a regulator of TNF-α mRNA stability and is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal. Using the THP-1 myelomonocytic cell line, we demonstrated for the first time that TTP is encoded by an mRNA with a short half-life under resting conditions. Using pharmacologic inhibitors of the mitogen-activated protein kinase pathways, we show that the induction of TTP by LPS activation is mediated through changes in transcription, mRNA stability, and translation. A coordinate increase in both TTP and TNF-α mRNA stability occurs within 15 min of LPS treatment, but is transduced through different mitogen-activated protein kinase pathways. This regulation of TTP and TNF-α mRNA stability is associated with the finding that TTP binds these mRNA under both resting and LPS-activated conditions in vivo. Finally, we demonstrate that TTP can regulate reporter gene expression in a TTP 3′ untranslated region-dependent manner and identify three distinct AU-rich elements necessary to mediate this effect. Thus, TTP regulates its own expression in a manner identical to that seen with the TNF-α 3′ untranslated region, indicating that this autoregulation is mediated at the level of mRNA stability. In this manner, TTP is able to limit the production of its own proteins as well as that of TNF-α and thus limit the response of the cell to LPS.

Structure/Function Analysis of Tristetraprolin (TTP): p38 Stress-Activated Protein Kinase and Lipopolysaccharide Stimulation Do Not Alter TTP Function

Tristetraprolin (TTP) is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal; however, the mechanism by which TTP mediated RNA instability is unknown. Using an established model system, we performed structure/function analysis with TTP as well as examined the current hypothesis that TTP function is regulated by p38-MAPKAP kinase 2 (MK2) activation. Deletion of either the N- or C-terminal domains inhibited TTP function. Extensive mutagenesis, up to 16%, of serines and threonines, some of which were predicted to mediate proteasomal targeting, did not alter human TTP function. Mutation of the conserved MK2 phosphorylation sites enhanced human TTP function in both resting and p38-stress-activated protein kinase-MK2-activated cells. However, p38-stress-activated protein kinase-MK2 activation did not alter the activity of either wild-type or mutant TTP. TTP localized to the stress granules, with arsenite treatment reducing this localization. In contrast, arsenite treatment enhanced stress granule localization of the MK2 mutant, consistent with the involvement of additional pathways regulating this event. Finally, we determined that, in response to LPS stimulation, human TTP moves onto the polysomes, and this movement occurs in the absence of 14-3-3. Taken together, these data indicate that, although p38 activation alters TTP entry into the stress granule, it does not alter TTP function. Moreover, the interaction of TTP with 14-3-3, which may limit entry into the stress granule, is not involved in the downstream message stabilization events.

Extracellular Signal-regulated Kinase Regulation of Tumor Necrosis Factor-α mRNA Nucleocytoplasmic Transport Requires TAP-NxT1 Binding and the AU-rich Element

Tumor necrosis factor-α (TNF-α) production is regulated by transcriptional and posttranscriptional mechanisms. Lipopolysaccharide activates the NFκB pathway increasing TNF-α transcription. Lipopolysaccharide also activates the mitogen-activated protein kinase pathways, resulting in stabilization and enhanced translation of the TNF-α message. In addition, nuclear export of the TNF-α mRNA is a posttranscriptionally regulated process involving the Tpl2-ERK pathway and requiring the presence of the TNF-α AU-rich element (ARE). We demonstrate that nuclear export of the TNF-α message requires not only the TNF-α ARE but also the interaction of the proteins TAP and NxT1, both of which are involved in nucleocytoplasmic transport of mRNA. Through the use of dominant negative ERK1 and ERK2, we establish that control of TNF-α mRNA nuclear export operates specifically through ERK1. Finally, we examined the role of two established TNF-α ARE-binding proteins, HuR and tristetraprolin, that shuttle between the nucleus and cytoplasm. These data demonstrate that neither tristetraprolin nor HuR is required for TNF-α mRNA export. It is unclear at this time if ARE-binding protein(s) directly interact with the TAP-NxT1 complex, if each complex is independently targeted by ERK1, or if only one complex is targeted.

Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

TNF-α is a central mediator of inflammation and critical for host response to infection and injury. TNF-α biosynthesis is controlled by transcriptional and posttranscriptional mechanisms allowing for rapid, transient production. Tristetraprolin (TTP) is an AU-rich element binding protein that regulates the stability of the TNF-α mRNA. Using a screen to identify TTP-interacting proteins, we identified Cullin 4B (Cul4B), a scaffolding component of the Cullin ring finger ligase family of ubiquitin E3 ligases. Short hairpin RNA knockdown of Cul4B results in a significant reduction in TNF-α protein and mRNA in LPS-stimulated mouse macrophage RAW264.7 cells as well as a reduction in TTP protein. TNF-α message t1/2 was reduced from 69 to 33 min in LPS-stimulated cells. TNF-3′ untranslated region luciferase assays utilizing wild-type and mutant TTP-AA (S52A, S178A) indicate that TTP function is enhanced in Cul4B short hairpin RNA cells. Importantly, the fold induction of TNF-α mRNA polysome loading in response to LPS stimulation is reduced by Cul4B knockdown. Cul4B is present on the polysomes and colocalizes with TTP to exosomes and processing bodies, which are sites of mRNA decay. We conclude that Cul4B licenses the TTP-containing TNF-α messenger ribonucleoprotein for loading onto polysomes, and reduction of Cul4B expression shunts the messenger ribonucleoproteins into the degradative pathway.

CARHSP1 is required for effective tumor necrosis factor alpha mRNA stabilization and localizes to processing bodies and exosomes.

ABSTRACT Tumor necrosis factor alpha (TNF-α) is a critical mediator of inflammation, and its production is tightly regulated, with control points operating at nearly every step of its biosynthesis. We sought to identify uncharacterized TNF-α 3′ untranslated region (3′UTR)-interacting proteins utilizing a novel screen, termed the RNA capture assay. We identified CARHSP1, a cold-shock domain-containing protein. Knockdown of CARHSP1 inhibits TNF-α protein production in lipopolysaccharide (LPS)-stimulated cells and reduces the level of TNF-α mRNA in both resting and LPS-stimulated cells. mRNA stability assays demonstrate that CARHSP1 knockdown decreases TNF-α mRNA stability from a half-life (t1/2) of 49 min to a t1/2 of 22 min in LPS-stimulated cells and from a t1/2 of 29 min to a t1/2 of 24 min in resting cells. Transfecting CARHSP1 into RAW264.7 cells results in an increase in TNF-α 3′UTR luciferase expression in resting cells and CARHSP1 knockdown LPS-stimulated cells. We examined the functional effect of inhibiting Akt, calcineurin, and protein phosphatase 2A and established that inhibition of Akt or calcineurin but not PP2A inhibits CARHSP1 function. Subcellular analysis establishes CARHSP1 as a cytoplasmic protein localizing to processing bodies and exosomes but not on translating mRNAs. We conclude CARHSP1 is a TNF-α mRNA stability enhancer required for effective TNF-α production, demonstrating the importance of both stabilization and destabilization pathways in regulating the TNF-α mRNA half-life.

Functional interactions between mRNA turnover and surveillance and the ubiquitin proteasome system.

The proteasome is a critical regulator of protein levels within the cell and is essential for maintaining homeostasis. A functional proteasome is required for effective mRNA surveillance and turnover. During transcription, the proteasome localizes to sites of DNA breaks, degrading RNA polymerase II and terminating transcription. For fully transcribed and processed messages, cytoplasmic surveillance is initiated with the pioneer round of translation. The proteasome is recruited to messages bearing premature termination codons, which trigger nonsense‐mediated decay (NMD), as well as messages lacking a termination codon, which trigger nonstop decay, to degrade the aberrant protein produced from these messages. A number of proteins involved in mRNA translation are regulated in part by proteasome‐mediated decay, including the initiation factors eIF4G, eIF4E, and eIF3a, and the poly(A)‐binding protein (PABP) interacting protein, Paip2. eIF4E‐BP (4E‐BP) is differentially regulated by the proteasome: truncated to generate a protein with higher eIF4B binding or completely degraded, depending on its phosphorylation status. Finally, a functional proteasome is required for AU‐rich‐element (ARE)‐mediated decay but the specific role the proteasome plays is unclear. There is data indicating the proteasome can bind to AREs, act as an endonuclease, and degrade ARE‐binding proteins. How these events interact with the 5′‐to‐3′ and 3′‐to‐5′ decay pathways is unclear at this time; however, data is provided indicating that proteasomes colocalize with Xrn1 and the exosome RNases Rrp44 and Rrp6 in untreated HeLa cells. Copyright

A Flexible Approach to Studying Post-Transcriptional Gene Regulation in Stably Transfected Mammalian Cells

The study of post-transcriptional regulation is constrained by the technical limitations associated with both transient and stable transfection of chimeric reporter plasmids examining the activity of 3′-UTR cis-acting elements. We report the adaptation of a commercially available system that enables consistent stable integration of chimeric reporter cDNA into a single genomic site in which transcription is induced by tetracycline. Using this system, we demonstrate the tight control afforded by this system and its suitability in mapping the regulatory function of defined cis-acting elements in the human TNF 3′-UTR, as well as the distinct effects of serum starvation on transiently transfected and stably integrated chimeric reporter genes.

Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity

Significance Nature produces a variety of proteins that could be tapped for therapeutic applications. This paper focuses on the bacterial enzyme β-lactamase, a component of antibody-directed enzyme prodrug therapies designed to activate cytotoxic prodrugs selectively at sites of malignancy. Unfortunately, like many other nonhuman proteins, β-lactamase evokes an antidrug immune response that limits its clinical potential. This paper demonstrates that a multiobjective library-design method enables incorporation of mutations throughout the protein, modifying portions that trigger immune recognition while simultaneously preserving stability and catalytic activity. The libraries were inherently enriched in beneficial variants, and they produced numerous candidates that were both highly functional and immunologically stealthy. The method is general purpose and could enable functional deimmunization of other biotherapeutic agents. Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide–MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.