Immune-activating cytokines such as interleukin-12 (IL-12) hold strong potential for cancer immunotherapy but have been limited by high systemic toxicities. We describe here an approach to safely harness cytokine biology for adoptive cell therapy through uniform and dose-controlled tethering onto the surface of the adoptively transferred cells. Tumor-specific T cells tethered with IL-12 showed superior antitumor efficacy across multiple cell therapy models compared to conventional systemic IL-12 coadministration. Mechanistically, the IL-12–tethered T cells supported a strong safety profile by driving interferon-γ production and adoptively transferred T cell activity preferentially in the tumor. Immune profiling revealed that the tethered IL-12 reshaped the suppressive tumor immune microenvironment, including triggering a pronounced repolarization of monocytic myeloid-derived suppressor cells into activated, inflammatory effector cells that further supported antitumor activity. This tethering approach thus holds strong promise for harnessing and directing potent immunomodulatory cytokines for cell therapies while limiting systemic toxicities.

Abstract Background: Interleukin-15 (IL-15) and interleukin-12 (IL-12) play complementary roles as immunomodulators. IL-15 induces T-cell memory and supports survival, activation, and proliferation of CD8 T and NK cells. IL-12 promotes T-cell cytotoxicity and innate immune responses in the tumor microenvironment (TME). Both cytokines have been explored as cancer immunotherapies, but severe side effects have limited clinical success. Torque has developed the Deep-PrimedTM T-cell therapy technology to direct the stimulatory activity of these cytokines to the TME, to prime and boost the immune response of T-cell therapies with limited systemic exposure and toxicity. Multitargeted T cells (MTC) specific for multiple tumor antigens are generated from patient apheresis. Cytokines are tethered to MTCs to support MTC persistence and activity following adoptive transfer into patients, while limiting systemic cytokine exposure. This study evaluates the combination of Deep IL-15 PrimedTM and Deep IL-12 PrimedTM T cells to leverage their complementary biology for superior efficacy. Methods: T cells reactive against MART-1 antigen were generated from healthy donors. Expansion and cytotoxicity of MART-1 T cells loaded with Deep IL-12, Deep IL-15, or both against cancer cells expressing MART-1 were assessed. Murine PMEL CD8 T cells reactive against the B16-F10 melanoma antigen gp100 were loaded with Deep IL-12, Deep IL-15, or both and evaluated for in vitro expansion, activation, and cytotoxicity against B16-F10 melanoma cells, as well as for antitumor activity in B16-F10 tumor-bearing mice. Results: Loading with Deep IL-15 promoted MART-1 T cell proliferation and preserved antigen reactivity over time. Deep IL-12 enhanced IFNγ secretion and cytotoxicity, particularly at low effector:target ratios. Combination of MART-1 T cells loaded with Deep IL-12 and Deep IL-15 further enhanced T-cell expansion, IFNγ secretion, and cytotoxicity. Similarly, combination of murine PMEL T cells loaded with Deep IL-12 and Deep IL-15 resulted in persistent T-cell activation, improved memory, and enhanced cytotoxicity over individually loaded T cells. Coadministration of Deep IL-12 and Deep IL-15 loaded PMEL T cells to B16-F10 melanoma-bearing mice resulted in increased IFN-γ and IP-10 systemic cytokine release, indicative of enhanced immune response. The treatment was well tolerated with minimal and reversible body weight loss, and no changes in clinical chemistry. Importantly, the combination of Deep IL-12 and Deep IL-15 elicited superior antitumor activity. Conclusions: Tethering DeepTM IL-12 and DeepTM IL-15 to T cells uniquely leverages their complementary functions as immunomodulators to maximize antitumor activity without notable toxicity in preclinical models. A phase I clinical trial of Deep IL-15 Primed MTCs (TRQ15-01) in solid cancers and lymphoma is enrolling (NCT03815682). Torque is initiating clinical evaluation of Deep IL-12 Primed MTCs (TRQ12-01), including in combination with TRQ15-01. Citation Format: Elena Geretti, Katharine Sackton, Pengpeng Cao, Shawn Carey, Xiaoyan Liang, Jonathan Nardozzi, Zishu Gui, Alicia Worthylake, Becker Hewes, Tap Maniar, Jonathan Fitzgerald, Andy Rakestraw, Douglas Jones, Karsten Sauer, Thomas Andresen. Combining Deep IL-12 PrimedTM and Deep IL-15 PrimedTM T cells induces potent antigen-dependent in vitro cytotoxicity and in vivo antitumor activity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A68.

Background Acquired resistance is a major limiting factor for durable T cell therapies in solid tumors. Antigen escape pathways such as insufficient antigen coverage or loss of target antigen remain major resistance mechanisms that need to be addressed in order to expand the field of T cell therapies.Interleukin-12 (IL-12) is a potent stimulator of innate and adaptive immune cells that holds strong potential for cancer immunotherapy, but its clinical utility has been limited by high systemic toxicities. We have previously shown that tethering an IL-12 immunocytokine to the surface of T cells prior to adoptive cell transfer (ACT) safely improves anti-tumor efficacy by promoting T cell function specifically in the tumor. Here, we demonstrate that cell-tethered IL-12 delivers adjuvant activity that leads to priming and expansion of by-stander, tumor-specific T cells, and thereby counteract common immune escape pathways. Methods Adjuvant activity of IL12-tethered pmel T cells, reactive towards the gp100 antigen of B16 tumors, was evaluated in the B16-OVA syngeneic mouse model. Notably, adoptive transfer of IL12-tethered pmel T cells, but not pmel T cells alone, resulted in proliferation of endogenous tumor infiltrating lymphocytes. To assess whether this reflected tumor-specific T cell responses, we used dextramer staining against non-targeted, tumor-specific antigens and found that both abundance and activation increased following cell-tethered IL-12 treatment. Encouraged by these findings, the OT-1 model was used to track epitope spreading to tumor-specific naïve T cells. Following treatment with IL-12-tethered PMEL T cells, we tracked the proliferation and tumor engraftment of labelled, naïve OT-I T cells, which are reactive towards the non-targeted OVA antigen. Results Cell-tethered-IL12, but neither ACT nor ACT and systemically administered IL-12, induced proliferation and engraftment OT-1 T cells in tumor-draining lymph nodes (tdLNs) and tumors of B16-OVA-bearing mice. This effect was antigen-dependent as the OT-I T cells were not primed in B16.F10 (OVA antigen-negative) tumors. Mechanistically, this priming was associated with IL-12-induced increases in activation and tdLN infiltration of cross-presenting dendritic cells (cDC1) as well as increased presentation of the SIINFEKL epitope of OVA specifically on this subset of dendritic cells. Conclusions Together, our findings suggest that tethering IL-12 to tumor-specific T cells prior to adoptive transfer promotes epitope spreading through the combination of tumor cell-killing induced by the ACT therapy and IL-12-induced activation of cDC1s in the tdLN. This adjuvant activity from T cell-tethered IL-12 holds promise for overcoming antigen escape pathways that limit the efficacy of antigen-specific T cells against heterogeneous tumors

Interleukin-12 (IL-12) is a potent pro-inflammatory cytokine that augments anti-tumor immune responses by promoting CD4 T cell Th1 differentiation, increasing CD8 T cell and NK cell cytotoxicity, inducing MHC expression on antigen presenting cells, and reprogramming myeloid-derived suppressor cells. However, the clinical utility of IL-12 has been limited by systemic toxicities. DeepTM IL-12 is a fusion protein between IL-12 and a Fab antibody against CD45, an abundant surface receptor on T cells. This enables tethering of IL-12 onto T cells prior to adoptive cell transfer (ACT). We report here the superior safety and efficacy of ACT with tumor-specific Deep IL- 12 Primed T cells which carry surface-tethered IL-12. The PMEL/B16-F10 cancer model utilizes adoptively transferred PMEL CD8 T cells that contain a TCR specific for the gp100 antigen expressed in B16-F10 melanoma cells. Deep IL-12 tethering to PMEL T cells significantly improved tumor growth inhibition and survival in tumor-bearing mice compared to PMEL T cell therapy alone or combined with systemically delivered IL-12. Doses up to 40 million Deep IL-12 Primed T cells did not result in overt toxicities. Thus, in contrast to systemically delivered free IL-12, T cell tethered IL-12 is well-tolerated. Deep IL-12 Primed PMEL T cells additionally resulted in elevated and sustained induction of IFNγ in the tumor microenvironment. In contrast, IFNγ induction in serum was transient and returned to baseline levels within four days of ACT. Further toxicological analyses will be discussed. In conclusion, ACT with Deep IL-12 Primed T cells delivers a favorable therapeutic profile by promoting T cell function selectively in the tumor microenvironment while limiting systemic IL-12 exposure.

Introduction Interleukin-12 (IL-12) is a potent cytokine that augments anti-tumor immune responses by promoting CD4 T cell Th1 differentiation, increasing CD8 T cell and NK cell cytotoxicity, inducing MHC expression on antigen presenting cells, and reprogramming myeloid-derived suppressor cells. However, the clinical utility of IL-12 administered systemically or produced by genetically engineered tumor-specific T cells has been limited by toxicities. DeepTM IL-12 is a fusion protein between IL-12 and a Fab antibody against CD45, an abundant surface receptor on T cells. This tethers IL-12 onto tumor-specific T cells prior to adoptive cell transfer (ACT) with the aim of reducing systemic exposure and focusing IL-12 function on the tumor microenvironment. Here, we describe the superior safety and efficacy profiles of Deep IL-12 tethered T cells in an immune competent adoptive cell therapy model for solid tumors. Methods The safety and efficacy of Deep IL-12 Primed T cells were evaluated in the PMEL/B16-F10 cancer model. CD8 PMEL T cells contain a TCR that recognizes the gp100 antigen expressed in B16-F10 melanoma cells. Deep IL-12 was loaded onto PMEL CD8 T cells to generate Deep IL-12 Primed PMEL T cells which were then adoptively transferred into mice bearing B16-F10 tumors. We compared tumor size, body weight change, cytokine release, circulating toxicity biomarkers, immune cell activity and histopathology in mice treated with Deep IL-12 Primed T cells and PMEL T cells alone or co-administered with recombinant IL-12. Results Deep IL-12 Primed PMEL T cells significantly improved tumor growth inhibition and overall survival in mice bearing established B16-F10 tumors as compared with PMEL T cell therapy alone or combined with systemic co-administration of IL-12. Deep IL-12 Primed PMEL T cells were well-tolerated, as administration of at least 40 million Deep IL-12 Primed PMEL T cells did not cause overt toxicities in non-tumor bearing mice. Deep IL-12 Primed PMEL T cells, but not PMEL T cells combined with systemic IL-12, resulted in elevated and sustained induction of IFNγ in the tumor microenvironment. In contrast, IFNγ induction in serum was transient and returned to baseline levels within four days of ACT. Interestingly, Deep IL-12 Priming increased both accumulation and activity of the adoptively transferred PMEL T cells in the tumor microenvironment but not in off-target healthy tissues such as the spleen. Further toxicological analysis of Deep IL-12 Primed tumor-reactive T cells will be discussed. Conclusions Deep IL-12 Priming enables tethering of IL-12 to the surface of tumor-specific T cells prior to ACT. This improves anti-tumor efficacy by promoting T cell function selectively in the tumor microenvironment and avoids toxicity by limiting systemic IL-12 exposure, resulting in a highly favorable benefit-risk profile for Deep IL-12 Primed T cells. Citation Format: De-Kuan Chang, Gulzar Ahmad, Jonathan Nardozzi, Katharine Sackton, Jesse Lyons, Karsten Sauer, Thomas Andresen, Douglas Jones. Surface-tethered IL-12 improves tumor-specific T-cell therapy and enhances inflammatory activity in tumors without inducing systemic toxicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 933.

Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD.

The proteins alpha-synuclein (αSyn) and leucine rich repeat kinase 2 (LRRK2) are both key players in the pathogenesis of the neurodegenerative disorder Parkinson's disease (PD), but establishing a functional link between the two proteins has proven elusive. Research studies for these two proteins have traditionally and justifiably focused in neuronal cells, but recent studies indicate that each protein could play a greater pathological role elsewhere. αSyn is expressed at high levels within neurons, but they also secrete the protein into the extracellular milieu, where it can have broad ranging effects in the nervous system and relevance to disease etiology. Similarly, low neuronal LRRK2 expression and activity suggests that LRRK2-related functions could be more relevant in cells with higher expression, such as brain-resident microglia. Microglia are monocytic immune cells that protect neurons from noxious stimuli, including pathological αSyn species, and microglial activation is believed to contribute to neuroinflammation and neuronal death in PD. Interestingly, both αSyn and LRRK2 can be linked to microglial function. Secreted αSyn can directly activate microglia, and can be taken up by microglia for clearance, while LRRK2 has been implicated in the intrinsic regulation of microglial activation and of lysosomal degradation processes. Based on these observations, the present review will focus on how PD-associated mutations in LRRK2 could potentially alter microglial biology with respect to neuronally secreted αSyn, resulting in cell dysfunction and neurodegeneration.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder, affecting 1–3% of the population over 65. Mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. The parkin protein possesses potent cell-protective properties and has been mechanistically linked to both the regulation of apoptosis and the turnover of damaged mitochondria. Here, we explored these two functions of parkin and the relative scale of these processes in various cell types. While biochemical analyses and subcellular fractionation were sufficient to observe robust parkin-dependent mitophagy in immortalized cells, higher resolution techniques appear to be required for primary culture systems. These approaches, however, did affirm a critical role for parkin in the regulation of apoptosis in primary cultured neurons and all other cells studied. Our prior work demonstrated that parkin-dependent ubiquitination of endogenous Bax inhibits its mitochondrial translocation and can account for the anti-apoptotic effects of parkin. Having found a central role for parkin in the regulation of apoptosis, we further investigated the parkin-Bax interaction. We observed that the BH3 domain of Bax is critical for its recognition by parkin, and identified two lysines that are crucial for parkin-dependent regulation of Bax translocation. Last, a disease-linked mutation in parkin failed to influence Bax translocation to mitochondria after apoptotic stress. Taken together, our data suggest that regulation of apoptosis by the inhibition of Bax translocation is a prevalent physiological function of parkin regardless of the kind of cell stress, preventing overt cell death and supporting cell viability during mitochondrial injury and repair.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and idiopathic Parkinson's disease. However, the mechanisms for activating its physiological function are not known, hindering identification of the biological role of endogenous LRRK2. The recent discovery that LRRK2 is highly expressed in cells of the innate immune system and genetic association is a risk factor for autoimmune disorders implies an important role for LRRK2 in pathology outside of the central nervous system. Thus, an examination of endogenous LRRK2 in immune cells could provide insight into the protein's function. Here, we establish that stimulation of specific Toll-like receptors results in a complex biochemical activation of endogenous LRRK2, with early phosphorylation of LRRK2 preceding its dimerization and membrane translocation. Membrane-associated LRRK2 co-localized to autophagosome membranes following either TLR4 stimulation or mTOR inhibition with rapamycin. Silencing of endogenous LRRK2 expression resulted in deficits in the induction of autophagy and clearance of a well-described macroautophagy substrate, demonstrating the critical role of endogenous LRRK2 in regulating autophagy. Inhibition of LRRK2 kinase activity also reduced autophagic degradation and suggested the importance of the kinase domain in the regulation of autophagy. Our results demonstrate a well-orchestrated series of biochemical events involved in the activation of LRRK2 important to its physiological function. With similarities observed across multiple cell types and stimuli, these findings are likely relevant in all cell types that natively express endogenous LRRK2, and provide insights into LRRK2 function and its role in human disease.

Missense mutation of the PARK8 gene, which encodes the protein Leucine‐Rich Repeat Kinase 2 (LRRK2), is the most common genetic cause of Parkinson's Disease (PD), yet the physiological role of this protein is largely unknown. LRRK2 dimerization has been linked to its activity and localization in the cell, as dimers have higher kinase activity and are preferentially membrane‐associated. In this study we utilized a Split‐Luciferase Protein‐ Fragment‐Assisted Complementation assay (PCA) to assess dimerization of LRRK2. PCA has numerous advantages over other techniques used to study protein‐protein interactions, as the luciferase reporter displays specificity, reversibility, and signal intensity. We have developed a quantitative bioluminescent assay of LRRK2 dimerization as a means to understanding the regulators of LRRK2 structure and function. Numerous split‐luciferase fragment pairs were explored from both the Renilla and Firefly luciferase reporters, and one fragment pair was optimized for investigating LRRK2 dimerization. Interestingly, we observed that fusion and orientation of luciferase fragments to their respective LRRK2 monomers were important factors for reporter efficiency. Efforts to characterize small molecule regulators of LRRK2 dimerization are ongoing.NIH grant #NS072604

Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of similar to 14-fold symmetry that catalyze the annealing of single-stranded DNAs in vitro. In this study, we investigated the structural elements that contribute to this distinctive higher order structural organization and examined its functional implications. A pair of vicinal cysteines, Cys-216 and Cys-217, was found to be essential for mtDNA maintenance. Mutations to the polar serine, the negatively charged aspartic and glutamic acids, and the hydrophobic amino acid alanine all destabilize mtDNA in vivo. The alanine mutants have an increased propensity of forming macroscopic filaments. In contrast, mutations to aspartic acid drastically destabilize the protein and result in unstructured aggregates with severely reduced DNA binding activity. Interestingly, the serine mutants partially disassemble the Mgm101 rings into smaller oligomers. In the case of the C216S mutant, a moderate increase in DNA binding activity was observed. By using small angle x-ray scattering analysis, we found that Mgm101 forms rings of similar to 200 angstrom diameter in solution, consistent with the structure previously established by transmission electron microscopy. We also found that the C216A/C217A double mutant tends to form broken rings, which likely provide free ends for seeding the growth of the super-stable but functionally defective filaments. Taken together, our data underscore the importance of a delicately maintained ring structure critical for Mgm101 activity. We discuss a potential role of Cys-216 and Cys-217 in regulating Mgm101 function and the repair of damaged mtDNA under stress conditions.

Homologous recombination (HR), an error free DNA repair mechanism, is poorly understood in mitochondria. This mechanism, evolutionarily conserved from bacteriophage to humans, has evolved primarily as a strategy for the repair of DNA double strand breaks. The recombinational repair of double strand DNA breaks may proceed by two distinct pathways. The conventional pathway requires a RecA/Rad51‐type recombinase that catalyzes strand invasion. The alternative pathway involves the Rad52‐type proteins, which promote recombination by single strand annealing. The Rad52‐type recombination proteins inherently form rings and filaments in vitro. The functional implications of these higher order structural organizations are not fully understood. We have recently shown that the mitochondrial genome maintenance protein, Mgm101, is a homologue of Rad52‐type recombination proteins. Like Rad52, Mgm101 forms rings. Loss of function mutations were identified that affect ring stability. Here, we show that the C‐tail, which is rich in conserved basic and aromatic residues, is unique to Mgm101 orthologues. We show that the residues K253, W257, R259, Y268 and the triple mutant K251A‐K253A are critical for function. Additionally, we show by size exclusion that these conserved residues fall into two categories: those that destabilize the ring structure of Mgm101 and those that do not. Our data support the idea that the Mgm101 C‐tail may represent a novel functional module for catalyzing DNA recombination. The biochemical characterization of these mutant proteins will shed light on the mechanism of mtDNA recombination. This work was supported by the grant R01AG023731 from the National Institute on Aging/NIH.

The human genome encodes six isoforms of importin alpha that show greater than 60% sequence similarity and remarkable substrate specificity. The isoform importin alpha 5 can bind phosphorylated cargos such as STAT1 and Epstein-Barr Virus Nuclear Antigen 1, as well as the influenza virus polymerase subunit PB2. In this work, we have studied the interaction of the nucleoporin Nup50 with importin alpha 5. We show that the first 47 residues of Nup50 bind to the C terminus of importin alpha 5 like a "clip," stabilizing the closed conformation of ARM 10. In vitro, Nup50 binds with high affinity either to empty importin alpha 5 or to a preassembled complex of importin alpha 5 bound to the C-terminal domain of the import cargo PB2, resulting in a trimeric complex. By contrast, PB2 can only bind with high affinity to importin alpha 5 in the absence of Nup50. This suggests that Nup50 primary function may not be to actively displace the import cargo from importin alpha 5 but rather to prevent cargo rebinding in preparation for recycling. This is the first evidence for a nucleoporin modulating the import reaction by directly altering the three-dimensional structure of an import adaptor.

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, has been shown to have opposite and often contradictory effects on the ability of cargos to be transported across the nuclear envelope. Without a clear connection between attachment of a phosphate moiety and biological response, it is difficult to fully understand and predict how phosphorylation regulates nucleocytoplasmic trafficking. In this review, we will recapitulate clue findings in the field and provide some general rules on how reversible phosphorylation can affect the nuclear-cytoplasmic localization of substrates. This is only now beginning to emerge as a key regulatory step in biology.

Interferon-gamma stimulation triggers tyrosine phosphorylation of the transcription factor STAT1 at position 701, which is associated with switching from carrier-independent nucleocytoplasmic shuttling to carrier-mediated nuclear import. Unlike most substrates that carry a classical nuclear localization signal (NLS) and bind to importin alpha 1, STAT1 possesses a nonclassical NLS recognized by the isoform importin alpha 5. In the present study, we have analyzed the mechanisms by which importin alpha 5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin alpha 5 with K-d = 191 +/- 20 nM. Whereas tyrosine phosphorylation at position 701 is essential to assemble a pSTAT1 importin alpha 5 complex, the phosphate moiety is not a direct binding determinant for importin alpha 5. In contrast to classical NLS substrates, pSTAT1 binding to importin alpha 5 is not displaced by the N-terminal importin beta binding domain and requires the importin alpha 5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin alpha 5 Armadillo (ARM) repeat 10 accompanies high-affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin alpha 5, which is inserted between ARM repeat 10 helices H1-H2-H3, thereby preventing intramolecular helical stacking essential to stabilize the folding conformation of ARM 10. Introducing a glycine at this position, as in importin alpha 1, disrupts high-affinity binding to pSTAT1, suggesting that pSTAT1 recognition is dependent on the intrinsic flexibility of ARM 10. Using the quantitative stoichiometry and binding data presented in this article, together with mutational information available in the literature, we propose that importin alpha 5 binds between two STAT1 monomers, with two major binding determinants in the SH2 and DNA binding domains. In vitro, this model is supported by the observation that a 38-mer DNA oligonucleotide containing two tandem cfosM67 promoters can displace importin alpha 5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus. (C) 2010 Elsevier Ltd. All rights reserved.

The Vaccinia virus H1 gene product, VH1, is a dual specificity phosphatase that down-regulates the cellular antiviral response by dephosphorylating STAT1. The crystal structure of VH1, determined at 1.32 Å resolution, reveals a novel dimeric quaternary structure, which exposes two active sites spaced ∼39 Å away from each other. VH1 forms a stable dimer via an extensive domain swap of the N-terminal helix (residues 1-20). In vitro, VH1 can dephosphorylate activated STAT1, in a reaction that is competed by the nuclear transport adapter importin α5. Interestingly, VH1 is inactive with respect to STAT1 bound to DNA, suggesting that the viral phosphatase acts predominantly on the cytoplasmic pool of activated STAT1. We propose that the dimeric quaternary structure of VH1 is essential for specific recognition of activated STAT1, which prevents its nuclear translocation, thus blocking interferon-γ signal transduction and antiviral response.

The rate of the alkaline phosphatase-catalyzed hydrolysis of 4-methylumbelliferone phosphate was measured in acoustically levitated droplets of aqueous tris (50 mM) at pH 8.5 at 22 +/- 2 degrees C and in supercooled solution at -6 +/- 2 degrees C. At 22 degrees C, the rate of product formation was in excellent agreement with the rate observed in bulk solution in a cuvette, indicating that the acoustic levitation process does not alter the enzyme activity. The rate of the reaction decreased 6-fold in supercooled solution at -6 +/- 2 degrees C. The acoustic levitator apparatus is described in detail.