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Deoxycytidine kinase (dCK) phosphorylates deoxycytidine, deoxyguanosine, and deoxyadenosine and plays an important role in the salvage pathway of nucleoside metabolism. dCK is also required for the phosphorylation of several antiviral and anticancer nucleoside drugs, with resistance to these agents often being associated with a loss or decrease in dCK activity. Data also indicate a role for dCK in immune function, and dCK inhibitors may provide treatment for immune disorders. To identify novel dCK inhibitors, the authors evaluated 2 existing biochemical assays, adapted both to high-throughput screening, and identified several series of hits. They also compared the potency of the hits between purified recombinant and endogenous enzyme. Meanwhile, they also developed a novel cell-based assay that rests on the rescue of cells from dCKdependent cytotoxic agents such as AraC. A large number of compounds were tested using the 3 assays, and a strong correlation in potency was observed between the biochemical assay using endogenous enzyme and the cell-based assay. The hits identified in these screens have proved to be good starting points for the synthesis of much more potent tool compounds to further investigate the physiological functions of dCK and potentially lead to the development of therapeutic agents. (Journal of Biomolecular Screening 2010:72-79)

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

In eukaryotic cells, the majority of proteins are degraded via the ATP-dependent ubiquitin/26S proteasome pathway. The proteasome is the proteolytic component of the pathway. It is a very large complex with a mass of around 2.5 MDa, consisting of at least 62 proteins encoded by 31 genes. The eukaryotic proteasome has evolved from a simpler archaebacterial form, similar in structure but containing only three different peptides. One of these peptides is an ATPase belonging to the AAA (Triple-A) family of ATPASES: Gene duplication and diversification has resulted in six paralogous ATPases being present in the eukaryotic proteasome. While sequence analysis studies clearly show that the six eukaryotic proteasomal ATPases have evolved from the single archaebacterial proteasomal ATPase, the deep node structures of the phylogenetic constructions lack resolution. Incorporating physical data to provide support for alternative phylogenetic hypotheses, we have constructed a model of a possible evolutionary history of the proteasomal ATPASES

We have isolated a cDNA clone from mouse, m56, that encodes a member of the Conserved ATPase-containing Domain (CAD) protein family. Sequence analysis revealed that m56 is identical to mouse mSug1/FZA-B and shares high homology with human Trip1, moth 18–56, and yeast Sug1. When examined, Sug1-like CAD proteins appear to function in the regulation of the 26S proteasome, as well as associate with members of the steroid/thyroid receptor superfamily and other transcriptional activators. m56 can complement the lethal phenotype of loss of SUG1 in yeast. We have examined the tissue distribution of m56 using Northern and Western blots, in addition to immunocytochemistry and in situ hybridization. While m56 was expressed in all tissues and cells examined, several classes of neurons, most notably in the hippocampus, olfactory bulb, and cerebellum, displayed elevated levels of m56 mRNA and protein. We also examined distribution of RNA polymerase II and 26S proteasome subunit 4 (S4) within the mouse brain by in situ hybridization. While all three genes had similar patterns of expression, there were significant differences among them. In moths, the expression of the Sug1 homolog 18–56 is dramatically up-regulated during programmed cell death. In addition, it has been previously demonstrated that the proteasome plays an essential role in the regulation of apoptosis in mammals. We examined the expression of m56 in mouse during natural and induced cell death in a variety of tissues and found no significant changes in expression. Taken together, the data presented here suggest that while m56 is a highly conserved gene that presumably plays essential but complex roles in basal and developmental processes, it may not represent a rate-limiting step in these processes. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 877–890, 1997

BIOCHEMICAL and genetic studies suggest the existence of mediators that work between the activation domains (ADs) of regulatory proteins and the basic transcriptional machinery. We have previously shown genetically that Sugl interacts with the AD of the yeast activator Ga141. Here we provide evidence that the Sugl protein of yeast binds directly to the ADs of Gal4 and the viral activator, VP16. Sugl protein is associated with the TATA-binding protein in vivo and binds to it in vitro, consistent with a mediator function. We also demonstrate that Sugl is not a component of the 26S proteasome, contrary to previous reports2–5. Sugl is a member of a large, highly conserved family of ATPases, implying a role for ATP hydrolysis in the activation of transcription.

PA700 is a 700,000-dalton multisubunit protein that activates multiple proteolytic activities of the 20 S proteasome by a mechanism dependent upon ATP hydrolysis (Ma, C.-P., Vu, J.H., Proske, R.J., Slaughter, C.A., and DeMartino, G.N. (1994) J. Biol. Chem. 269, 3539-3547). In order to determine the identities of and structural relationships among the subunits of PA700, individual PA700 subunits were isolated by a combination of reverse phase high performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis. Seven of the 16 subunits of PA700 so isolated were subjected to solid phase protease digestion followed by reverse phase HPLC. Selected peptides from each protein were sequenced by automated Edman degradation. Comparison of the resulting amino acid sequences with those in current data bases indicated that three of the subunits represented novel proteins, whereas four subunits were homologous to previously describe proteins. Three subunits of the latter group were, in turn, homologous to one another and are members of a large family of proteins containing a consensus sequence for ATP binding. Purified PA700 demonstrated ATPase activity. Treatment of PA700 with alkylating agents, such as N-ethylmaleimide, inhibited with similar kinetics both proteasome activation and ATPase activity, suggesting that these two activities are functionally linked. Thus, PA700 is composed of multiple members of a protein family that may function in the ATP-dependent regulation of proteasome activity.

BACKGROUND: S1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments. METHODOLOGY: In order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models. PRINCIPAL FINDINGS: Our analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL(-/-)) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PL(H/-)) or two alleles (S1PL(H/H)) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL(-/-) mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation. SIGNIFICANCE: These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs.

The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.

The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins.

We have identified a novel protein, CADp44, based on the analysis of cDNAs derived from the brainstem of the 13-lined ground squirrel, Spermophilus tridecemlineatus. CADp44 has an unmodified molecular mass of 44 178 Da and contains multiple functional domains, including a conserved ATPase domain (CAD) and a leucine zipper motif. We show that distinct regions of the CADp44 sequence are identical to a set of peptides prepared from a recently identified bovine protein, referred to as p42, which is found in the PA700 regulatory complex of the 26S proteasome (DeMartino et al., 1996). We also show that CADp44 is the functional homolog of the newly characterized Sug2 protein from the budding yeast, Saccharomyces cerevisiae (Russell et al., 1996). Consistent with its role as a component of the 26S proteasome, CADp44 mRNA is found in all ground squirrel tissues examined. Evolutionary relationships based on sequence analysis show that both CADp44 and yeast Sug2p are distinct from the other five CAD ATPases found in the PA700, and together comprise the sixth and newest CAD subunit of the regulatory complex of the 26S proteasome.

This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding.

THE thyroid-hormone receptors are hormone-dependent transcription factors that control expression of many target genes1,2. This regulation is presumably a consequence of hormone-dependent contacts between the receptors and the basal transcription machinery3. We used the yeast two-hybrid system4,5 to identify a candidate human transcriptional mediator that interacts with both the thyroid-hormone receptor and the retinoid-X receptor in a ligand-dependent fashion. This protein, Tripl (for thyroid-hormone-receptor interacting protein), shares striking sequence conservation with the yeast transcriptional mediator Sugl (refs 6, 7). Here we show that Tripl can functionally substitute for Sugl in yeast, and that both proteins interact in vitro with the thyroid-hormone receptor, and with the transcriptional activation domains of yeast GAL4 and of herpes virus VP16.

Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis. Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae. The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase). Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced). The TRP3pr gene was able to complement a trp3:: URA3 null mutation in yeast. No significant differences in the doubling times of TRP3 or TRP3pr yeast transformants were observed during growth at 25 degrees C, 30 degrees C or 37 degrees C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan. However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA. This observation which is statistically significant (P0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein.

THE acidic transcriptional activation motif functions in all eukary-otes1–4, which suggests that it makes contact with some universal component of the transcriptional apparatus. Transcriptional activation by the yeast regulatory protein GAL4 requires an acidic region at its carboxyl terminus. Here we implement a selection scheme to determine whether GAL4 can still function when this C-terminal domain has been deleted. It can, when accompanied by a mutation in the SUG1gene which is an essential gene in yeast. Analysis of mutant SUG1in combination with various alleles of GAL4 indicates that SUG1 acts through a transcriptional pathway that depends on GAL4, but requires a region of GAL4 other than the C-terminal acidic activation domain. The predicted amino-acid sequence of SUG1 closely resembles that of two human proteins, TBP1 and MSS1, which modulate expression mediated by the human immunodeficiency virus tatgene.

The ceramide metabolite sphingosine 1-phosphate (S1P) is an important modulator of a wide variety of physiologic functions via its action as a second messenger in intracellular signaling pathways and its extracellular interaction with specific G protein-coupled receptors on the cell surface. In the immune system, interaction of S1P with one of its receptors, S1P1, leads to inhibition of lymphocyte egress from primary and secondary lymphoid tissues resulting in peripheral lymphopenia, a mechanism with demonstrated effects in recent human clinical studies using the S1P1 receptor agonist, FTY720. S1P lyase (S1PL), which catalyzes the only irreversible step in sphingosine metabolism, the degradation of S1P to hexadecenal and ethanolamine phosphate, has recently been suggested to be a target of 2-acetyl-4-tetrahydroxybutylimidazole (THI), a compound known to increase tissue S1P levels and alter lymphocyte trafficking. In the current study, genetic inactivation of S1PL in knockout (KO) mice resulted in severe depletion of lymphocytes in the peripheral blood accompanied by accumulation of mature T cells in the thymus. This abnormal pattern of lymphocyte distribution suggests that results previously seen in THI treated mice were indeed due to THI’s inhibition of S1PL rather than another unknown activity. S1PL KO mice also demonstrated significantly reduced viability, perhaps due to increased numbers of myeloid cells which infiltrated the lungs. In a S1PL humanized allele, in which the human S1PL has been introduced into S1PL KO mice and restores ∼17% of wild type S1PL activity, the reduced viability trait is rescued while peripheral lymphopenia is maintained. Since the phenotypes are genetically separable by an allele that reduces S1PL activity, the lack of observed toxicity at doses of THI resulting in lymphopenia is likely due to its partial inhibition of S1PL activity at those doses, perhaps due to differences in S1P or S1P receptor expression or metabolism in different cell types. The potential for separation of lymphopenia and myeloid cell lung pathology effects by chemical inhibition was confirmed by chronic treatment of mice with the oral, small molecule S1PL inhibitor, LX2931, for 47 days during which no effects on viability were observed while the altered profile of lymphocyte migration seen in the partially inhibited humanized S1PL allele was maintained. Rats and monkeys treated with LX2931 also exhibited peripheral lymphopenia suggesting that the S1PL signaling pathway is conserved across mammalian species. Both S1PL KO and LX2931 treated mice were protected in the oxazolone challenge assay of delayed type hypersensitivity and the collagen induced model of rheumatoid arthritis, demonstrating that the reduction of peripheral lymphocytes previously observed with S1PL inhibition can beneficially affect the development of autoimmune disease.