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The quest to elucidate the molecular mechanism of action of rapamycin in the early 1990s led to the discovery of the novel TOR (target of rapamycin) proteins in yeast and mammalian cells. This was a major breakthrough that resulted in the development of new rapamycin analogs as anti-cancer agents, and launched new research that revealed the pre-eminent biological role of mTOR (mammalian or mechanistic TOR). Beyond mediating rapamycin sensitivity, the TOR proteins are nutrient sensing protein kinases, conserved from yeast to man, with a core function in regulating cell growth, metabolism and overall cell survival. There have been many insightful historical accounts of the origins of TOR; however, the complete TOR dossier would benefit from a chapter on the untold story of the simultaneous co-discovery of the yeast TOR proteins by two independent laboratories, one that is inclusive of the discoveries made at the former SmithKline Beecham (legacy GlaxoSmithKline). Accordingly, this comprehensive retrospective retraces the provenance of yeast TOR (circa 1990-1996) and highlights the early groundbreaking publications that revealed the identity of the TOR genes and proteins. It also commemorates key companion papers which helped to clarify yeast TOR gene nomenclature, identified structural motifs in the predicted TOR protein sequences, demonstrated interactions between yeast FKBP12-rapamycin and TOR, characterized mutations responsible for drug resistance, and began to decipher TOR protein function; some of these crucial early studies appeared in this journal (e.g., Koser et al., 1993. Gene 129, 159-165; Cafferkey et al., 1994. Gene 141, 133-136; Freeman and Livi, 1996. Gene 172, 143-147). A period of intensive investigation, events are portrayed chronologically and juxtaposed alongside the independent parallel efforts to identify and purify mTOR. Finally, in a broader historical context, TOR and mTOR are examined a posteriori as paragons of multiple discovery, illustrating how this common phenomenon (also known as simultaneous invention) can greatly accelerate problem solving and advance human knowledge in a fast-breaking area of scientific research.

Background The amino acid response ( AAR ) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti‐inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl‐ tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. Methods and Results Consistent with its ability to inhibit prolyl‐ tRNA synthetase, halofuginone elicited a general control nonderepressible 2–dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l ‐proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II /phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2–dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell–derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin‐1‐mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/ eIF 2α‐dependent manner. Conclusions Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

The analysis of pharmaceutical industry data indicates that the major reason for drug candidates failing in late stage clinical development is lack of efficacy, with a high proportion of these due to erroneous hypotheses about target to disease linkage. More than ever, there is a requirement to better understand potential new drug targets and their role in disease biology in order to reduce attrition in drug development. Genome editing technology enables precise modification of individual protein coding genes, as well as noncoding regulatory sequences, enabling the elucidation of functional effects in human disease relevant cellular systems. Areas covered: This article outlines applications of CRISPR genome editing technology in target identification and target validation studies. Expert opinion: Applications of CRISPR technology in target validation studies are in evidence and gaining momentum. Whilst technical challenges remain, we are on the cusp of CRISPR being applied in complex cell systems such as iPS derived differentiated cells and stem cell derived organoids. In the meantime, our experience to date suggests that precise genome editing of putative targets in primary cell systems is possible, offering more human disease relevant systems than conventional cell lines.

The T cell receptor (TCR), a defining structure of T cells is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an immune response. The extreme diversity of the TCR repertoire represents a major analytical challenge. Massively parallel high throughput sequencing is commonly deployed to characterize various key features of T cell receptors including their clonal expansion and repertoire diversity. Genomic DNA is the most widely employed starting material to characterize the TCR repertoire. DNA is highly stable and contains a fixed copy number per cell, which allows for better quantification of TCR clones. However, the inclusiveness is a major limitation of using genomic DNA along with the PCR amplification challenges and sequencing errors due to recombined gene segments within the context of ‘unused’ segments and introns. Here we deploy RNA-based TCR profiling as an alternate strategy to overcome the major limitations associated with the DNA-based approach. Employing RNA with a simplified PCR amplification strategy allows a more comprehensive identification of unique TCR variants with greater sensitivity. It also provides information about expression levels of TCR sequences, and more importantly, excludes the non-productive sequences that are functionally irrelevant. In addition, Unique Molecular Indexing (UMIs) enables the determination of the absolute count of RNA transcripts processed in a sample, allowing straightforward error corrections. Citation Format: Shanker Kalyana-Sundaram, Christopher Traini, Wendy Halsey, Heather Jackson, Sabyasachi Bhattacharya, Niranjan Yanamandra, Ganesh Sathe, George Livi. Comparative analysis between DNA vs RNA-based T cell repertoire profiling for cancer immunotherapy [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 1208.

Elevated expression of the c-MYC oncogene (either due to gene amplification, translocation, abnormality in upstream signaling pathways and/or protein stabilization) is one of the most common abnormalities in human cancers. Efforts to identify direct pharmacological inhibitors of c-MYC function have not yet yielded drug-like molecules. Therefore, we sought to pursue alternative screening strategies for this classically “undruggable” transcription factor. We developed a novel antibody-based high-throughput HTRF screening assay that specifically detects endogenous c-MYC protein levels in a MYC amplified cancer cell line. Taking advantage of the short half-life of c-MYC, both at the protein and mRNA level, we conducted a cell-based screen of the GSK screening collection to identify compounds that can rapidly decrease c-MYC protein levels. Elimination of false positive hits using stringent triage assays successfully identified two valid hit series exemplified by GSK970 and GSK417. Molecular mode of action studies revealed these molecules inhibit MYC transcription by binding to the minor groove of DNA with AT sequence specificity. Unfortunately, this mechanism of c-MYC inhibition demonstrated poor in vivo translatability as tissue DNA acts as a molecular sink, effectively sequestering compound, and limiting its pharmacodnyamic response. Citation Format: Biju Mangatt, Anthony D. Pietra, Anna Waszkiewicz, Jon-Paul Jaworski, Sonja Ghidelli-Disse, Thomas J. Berrodin, Christian S. Sherk, Derrick W. Meinhold, Anna Rutkowska-Klute, Shanker K. Sundaram, Gopinath Ganji, Wendy S. Halsey, George P. Livi, William Li, James Mack, Stuart P. Romeril, Elisabeth A. Minthorn, Rakesh Kumar, Gerard C. Drewes, Dirk A. Heerding, Lorena A. Kallal, Carolyn A. Buser, Jesus R. Medina. Discovery of small-molecule compounds targeting c-MYC using a novel cell-based screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2919.

During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.

The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli. Once in the cytosol, HtrA2/Omi has been implicated in promoting cell death by binding to inhibitor of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif, thus inducing caspase activity, and also in mediating caspase-independent death through its own protease activity. We report here the phenotype of mice entirely lacking expression of HtrA2/Omi due to targeted deletion of its gene, Prss25. These animals, or cells derived from them, show no evidence of reduced rates of cell death but on the contrary suffer loss of a population of neurons in the striatum, resulting in a neurodegenerative disorder with a parkinsonian phenotype that leads to death of the mice around 30 days after birth. The phenotype of these mice suggests that it is the protease function of this protein and not its IAP binding motif that is critical. This conclusion is reinforced by the finding that simultaneous deletion of the other major IAP binding protein, Smac/DIABLO, does not obviously alter the phenotype of HtrA2/Omi knockout mice or cells derived from them. Mammalian HtrA2/Omi is therefore likely to function in vivo in a manner similar to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress, and not like the proapoptotic Reaper family proteins in Drosophila melanogaster.

GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales , and increases in others, the Betaproteobacteria , Gammaproteobacteria , and Bifidobacteriaceae . Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.)

We report the identification and characterization of a novel cytokine-like gene family using structure-based methods to search for novel four-helix-bundle cytokines in genomics databases. There are four genes in this family, FAM3A, FAM3B, FAM3C, and FAM3D, each encoding a protein (224–235 amino acids) with a hydrophobic leader sequence. Northern analysis indicates that FAM3B is highly expressed in pancreas, FAM3D in placenta, and FAM3A and FAM3C in almost all tissues. Immunohistochemistry showed that FAM3A is expressed prominently in the vascular endothelium, particularly capillaries. We found that FAM3A and FAM3B protein were both localized to the islets of Langerhans of the endocrine pancreas. Recombinant FAM3B protein has delayed effects on β-cell function, inhibiting basal insulin secretion from a β-cell line in a dose-dependent manner.

The enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyses the first step in aromatic amino acid biosynthesis in prokaryotes, plants and fungi. Cells of Saccharomyces cerevisiae contain two catalytically redundant DAHP synthases, encoded by the genes ARO3 and ARO4, whose activities are feedback-inhibited by phenylalanine and tyrosine, respectively. ARO3/4 gene transcription is controlled by GCN4. The authors previously cloned an ARO3 gene orthologue from Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine, and (2) a homozygous aro3-deletion mutant of C. albicans is phenotypically Aro+, suggesting the existence of another isozyme(s). They now report the identification and functional characterization of the C. albicans orthologue of S. cerevisiae Aro4p. The two Aro4p enzymes share 68% amino acid identity. Phylogenetic analysis places the fungal DAHP synthases in a cluster separate from prokaryotic orthologues and suggests that ARO3 and ARO4 arose from a single gene via a gene duplication event early in fungal evolution. C. albicans ARO4 mRNA is elevated upon amino acid starvation, consistent with the presence of three putative Gcn4p-responsive elements (GCREs) in the gene promoter sequence. C. albicans ARO4 complements an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess tyrosine. The authors engineered Δaro3/Δaro3 Δaro4/MET3p::ARO4 cells of C. albicans (with one wild-type copy of ARO4 placed under control of the repressible MET3 promoter) and found that they fail to grow in the absence of aromatic amino acids when ARO4 expression is repressed, and that this growth defect can be partially rescued by aromatic amino acids and certain aromatic amino acid pathway intermediates. It is concluded that, like S. cerevisiae, C. albicans contains two DAHP synthases required for the first step in the aromatic amino acid biosynthetic pathway.

Importance Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients. Objective To characterize the transcriptomics of peripheral monocytes in patients with ALS. Design, Setting, and Participants Monocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction. Main Outcomes and Measures The differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling. Results The demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% were men and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B , IL8 , FOSB , CXCL1 , and CXCL2 , were confirmed by quantitative reverse transcription polymerase chain reaction ( IL8 , mean [SE], 1.00 [0.18]; P = .002; FOSB , 1.00 [0.21]; P = .009; CXCL1 , 1.00 [0.14]; P = .002; and CXCL2 , 1.00 [0.11]; P = .01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients. Conclusions and Relevance Our data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.

GSK2251052, a novel leucyl-tRNA synthetase (LeuRS) inhibitor, was in development for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. In a phase II study (study LRS114688) evaluating the efficacy of GSK2251052 in complicated urinary tract infections, resistance developed very rapidly in 3 of 14 subjects enrolled, with ≥32-fold increases in the GSK2251052 MIC of the infecting pathogen being detected. A fourth subject did not exhibit the development of resistance in the baseline pathogen but posttherapy did present with a different pathogen resistant to GSK2251052. Whole-genome DNA sequencing of Escherichia coli isolates collected longitudinally from two study LRS114688 subjects confirmed that GSK2251052 resistance was due to specific mutations, selected on the first day of therapy, in the LeuRS editing domain. Phylogenetic analysis strongly suggested that resistant Escherichia coli isolates resulted from clonal expansion of baseline susceptible strains. This resistance development likely resulted from the confluence of multiple factors, of which only some can be assessed preclinically. Our study shows the challenges of developing antibiotics and the importance of clinical studies to evaluate their effect on disease pathogenesis. (These studies have been registered at ClinicalTrials.gov under registration no. NCT01381549 for the study of complicated urinary tract infections and registration no. NCT01381562 for the study of complicated intra-abdominal infections.)

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.

Initial steps in investigating gene function often include deleting and overexpressing the gene of interest and identifying the subcellular location of the gene product. To facilitate these procedures, a number of new PCR modules, which contain selectable markers and in some cases other genetic elements (e.g promoter elements, epitope tags, and reporter genes) have been developed. These modules are used as PCR substrates to create products that can be targeted to specified locations in the yeast genome, thus modifying that genomic locus. We describe here a series of plasmids that contain a truncated version of the strong ADH1 promoter with and without amino-terminal 3HA and GST tags. Because these plasmids contain the same vector sequences as the GAL1 promoter plasmids, a constitutive and an inducible promoter can now be integrated with a minimal number of primers.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 “decoy” receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the “decoy” receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 °C, their rank-ordered affinities are substantially different at 37 °C, with DR5 having the highest affinity (K D ≤ 2 nm) and OPG having the weakest (K D = 400 nm). Preferentially enhanced binding of TRAIL to DR5 was also observed at the cell surface. These results reveal that the rank ordering of affinities for protein-protein interactions in general can be a strong function of temperature, and indicate that sizeable, but hitherto unobserved, TRAIL affinity differences exist at physiological temperature, and should be taken into account in order to understand the complex physiological and/or pathological roles of TRAIL.

The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells. Both forms of recombinant Yak1p kinase were catalytically active and preferred myelin basic protein (MBP) as a substrate over several other proteins. Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164. Peptides containing each site were obtained and tested as Yak1p substrates. Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP. The maximal velocity (Vmax) values of the full-length Yak1p reaction were 110±21 (Ser-164) and 8.7±1.7 (MBP), and those of N-terminally truncated Yak1p were 560.7±74.8 (Ser-164) and 34.4±2.2 (MBP) pmol/min per mg of protein. Although neither form of Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro. Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity. Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest that Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1–536) as well as four other cDNAs (nt 1459–1632, nt 1765–1986, nt 2152–2538, and nt 2978–3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.

The TOR genes were first identified in Saccharomyces cerevisiae by the isolation of mutants which exhibit dominant resistance to the immunosuppressive and antifungal drug rapamycin (Rm). The originally characterized Rm-resistant (Rm R ) TOR1 -1 and TOR2-1 alleles contain an Arg in place of a conserved Ser residue, which lies adjacent to the phosphatidylinositol (PI) kinase-related domain of TOR (Ser 1972 in TOR1; Ser 1975 in TOR2). Additional spontaneous Rm R mutants containing Lys, Ile or Asn substitutions were subsequently isolated. As this Ser is a potential site for protein kinase C phosphorylation, we were interested in determining whether the observed Rm R is due to steric hindrance of the FKBP12-Rm-TOR interaction or whether phosphorylation at this site is required to mediate the interaction. Using site-directed mutagenesis, we replaced the Ser 1972 residue of TOR1 with either a conservative residue, Ala, an alternative potential phosphorylation site, Thr, or Asp to mimic phosphorylation. The TOR1(S1972A) mutant protein retained Rm sensitivity (Rm S ), whereas both the Thr and Asp substitutions conferred Rm R . Rm S correlated with the ability to interact with FKBP12-Rm in a two-hybrid assay: both wild-type TOR1 and the S1972A mutant retained the ability to interact with FKBP12-Rm, whereas the S1972T, S1972D and S1972R mutants failed to interact. All mutant TOR1 proteins were able to complement the growth defect of torl null alleles, suggesting that the Ser 1972 residue may not be required for TOR1 function in cycling cells. Since a TOR1(S1972A) mutant protein confers a Rm s phenotype, interacts with FKBP12-Rm in a two-hybrid assay, and functions in vivo, we conclude that phosphorylation at Ser 1972 is not necessary for the interaction between TOR1 and FKBP12-Rm.

CSBP1 and CSBP2 are human homologues of the Saccharomyces cerevisiae Hog1 mitogen-activated protein kinase which is required for growth in high osmolarity media. Expression of CSBP1, but not CSBP2, complemented a hog1 delta phenotype. A CSBP2 mutant (A34V) that complements hog1 delta was isolated and found to have approximately 3-fold lower kinase activity than the wild-type CSBP2. Further analysis revealed that both the kinase activity and tyrosine phosphorylation of CSBP1 and CSBP2 (A34V) is regulated by salt. In contrast, wild-type CSBP2 is constitutively active but dependent on the upstream kinase, Pbs2. Mutagenesis studies showed that reduction or elimination of CSBP2 kinase activity restores salt responsiveness as measured by tyrosine phosphorylation suggesting that too high a level of kinase activity can result in desensitization of the host cell and inability to grow in high salt.

Recombinant baculoviruses were constructed to express cDNAs encoding two distinct subtypes of human cAMP-specific phosphodiesterase (hPDE4A and hPDE4B). Infection of Spodoptera frugiperda insect cells with the appropriate recombinant baculoviruses resulted in high level production of biologically-active protein as measured by enzymatic activity and immunoblotting using subtype-specific anti-hPDE4 antisera. Both recombinant proteins showed catalytic activity with a low Km (approximately 3 microM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent Kis of 0.38 and 0.25 microM, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (Kd approximately 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.

Transfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMI 1640 (RPMI) at 37 degrees C and 5% CO2 causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal-specific genes as well as genes expressed in response to changes in temperature, CO2 and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)- and hyphal (RPMI)-specific probes resulting in identification of a gene expressed in response to culture conditions but not regulated by the yeast-hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiae alpha-agglutinin, encoded by AG alpha 1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C. albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for agglutinin-like sequence). While the N- and C-termini of the predicted 1260-amino-acid ALS1 protein resemble those of the 650-amino-acid AG alpha 1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36-amino-acid sequence not present in AG alpha 1. These repeats are also present on the nucleotide level as a highly conserved 108 bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and, in some cases, between ALS1 alleles in the same strain, suggesting a strain-dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.

The control of amino acid and purine biosynthesis in the yeast Saccharomyces cerevisiae is mediated by the transcriptional activator GCN4. We previously identified the presence of two putative GCN4 responsive elements (GCREs) in the promoter sequence of the Candida albicans ARO3 gene, which encodes an enzyme in the aromatic amino acid pathway. We now show that amino acid deprivation results in a dramatic rise in the steady-state level of ARO3-specific mRNA, indicative of a GCN-like pathway in C. albicans.

Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.

The yeast TOR1 and TOR2 proteins were previously discovered as putative targets of the immunosuppressive drug rapamycin. Although their cellular function is unknown, they are predicted to be at least 215 kDa in size and possess a C-terminal phosphatidylinositol (PI) kinase-related domain. We previously identified a conserved Ser residue, within the PI kinase-related domain of both yeast TOR proteins (Ser1972 in TOR1; Ser1975 in TOR2), as being the site of missense mutations conferring dominant rapamycin resistance. The Ser1972/1975 residue of yeast TOR is conserved in mammalian TOR homologs. One possibility is that this residue is critical for a direct interaction between TOR and the FKBP12-rapamycin complex. There is very recent biochemical evidence for an interaction between mammalian TOR and FKBP12-rapamycin (Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994) Nature 369, 756-758; Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. (1994) Cell 78, 35-43). Using the yeast two-hybrid system, we now have obtained genetic proof of a physical interaction between FKBP12-rapamycin and TOR and have demonstrated that this interaction requires the conserved Ser residue. We have found that a small fragment of wild-type yeast TOR2 spanning Ser1975 is capable of interacting with human FKBP12 in the presence of rapamycin, whereas an Arg1975 mutant fails to interact. This effect is dependent upon rapamycin and is antagonized by FK506.

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

We have isolated cDNA clones from human frontal cortex cDNA libraries that encode a unique subtype of the low-Km, cAMP-specific phosphodiesterases (PDEs IV). The 564-amino acid sequence of the protein (human brain PDE IV (hPDE IVB)) shows significant homology to a PDE IV subtype expressed in human monocytes (hPDE IVA), particularly within the approximately 300-amino acid PDE IV catalytic domain. The degree of protein sequence identity is much greater between hPDE IVB and a homolog derived from rat brain (92% over 562 amino acids) than between hPDE IVB and hPDE IVA (76% over 538 amino acids), suggesting a greater subtype-specific versus species-specific conservation of protein sequence. Analysis of the distribution of hPDE IVB mRNA expression revealed a restricted pattern, with an approximately 4-kilobase mRNA detected in brain, heart, lung, and skeletal muscle and not in placenta, liver, kidney, or pancreas. An additional approximately 5-kilobase hPDE IVB-related mRNA species was detected in brain tissue. Recombinant hPDE IVB displayed all of the expected kinetic characteristics for a PDE IV, including sensitivity to the isozyme-selective inhibitor rolipram (Ki = 0.085 microM). Scatchard analysis of (R)-[3H]rolipram binding data suggested the presence of two noninteracting high affinity rolipram-binding sites (Kd = 0.4 and 6 nM) or a negatively cooperative interaction among multiple binding sites.

The four earliest developmentally controlled enzymes in the cellular slime mold, Dictyostelium discoideum, accumulate during axenic growth in rich media. We have shown that at low cell titers the specific activities of N-acetylglucosaminidase, alpha-mannosidase, leucine aminopeptidase, and alanine transaminase are each at very low or vegetative levels comparable to amoebae which have been grown on bacteria as the food source. During the exponential phase of growth all four enzymes accumulate dramatically reaching cellular specific activities at least as high as during development. The magnitude of this accumulation is influenced by alterations in the growth medium. We suggest that these results, combined with those of prior investigations, indicate that a restricted segment of early development is initiated during axenic growth. This means that growth and early development are not mutually exclusive events in this organism. The secretion of lysosomal enzymes is also affected by the composition of the growth media. In all media, including growth in bacterial suspensions, lysosomal enzymes are secreted in significant quantities. There is a correspondence in the effects of media composition on the secretion of these enzymes and on the regulation of developmentally controlled enzymes during axenic growth. The secretion of lysosomal enzymes that are not developmentally regulated is affected in these media, suggesting that the regulation and secretion of these enzymes are under separate control. It is clear that studies of the regulation of lysosomal enzymes in this organism must take into account the secretion of the enzymes as well as their cellular specific activities to properly reflect levels of gene expression.

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.

P2Y receptors have been reported to modulate gastrointestinal functions. The newest family member is the nucleotide-sugar receptor P2Y14. P2ry14 mRNA was detected throughout the rat gut, with the highest level being in the forestomach. We investigated the role of the receptor in stomach motility using cognate agonists and knockout (KO) mice. In rat isolated forestomach, 100 μM UDP-glucose and 100 μM UDP-galactose both increased the baseline muscle tension (BMT) by 6.2 ± 0.6 and 1.6 ± 0.6 mN ( P < 0.05, n = 3–4), respectively, and the amplitude of contractions during electrical field stimulation (EFS) by 3.7 ± 1.7 and 4.3 ± 2.5 mN ( P < 0.05, n = 3–4), respectively. In forestomach from wild-type (WT) mice, 100 μM UDP-glucose increased the BMT by 1.0 ± 0.1 mN ( P 14 receptor associated with contractility in the rodent stomach that does not lead to altered gastric emptying after receptor deletion and an ability of UDP-glucose to delay gastric emptying without involving the P2Y14 receptor.

Cyclophilin (CyP), a major cytosolic protein possessing peptidyl-prolyl cis-trans isomerase activity, has been implicated as the specific receptor of the immunosuppressive drug cyclosporin A (CsA). To identify other potential CsA receptors related to CyP, two human cDNA libraries were screened under low stringency conditions using human CyP cDNA (encoding hCyP1) as a probe. Two cDNAs were identified which encode distinct proteins related to human hCyP1. These two novel proteins, designated hCyP2 and hCyP3, share 65 and 76% amino acid sequence homology with hCyP1, respectively. Both hCyP2 and hCyP3 contain NH2-terminal hydrophobic extensions of 32 and 42 amino acids, respectively. Protein-specific antibodies revealed the predominant association of hCyP2 and hCyP3 with membranes and subcellular organelles, which suggests that the amino-terminal leader sequences of the two CyP isoforms may act as signal peptides. In contrast to the results with hCyP1, Southern blot analysis indicated that both hCyP2 and hCyP3 gene sequences are represented infrequently in the human genome. Northern and Western blot analysis showed that the distribution of mRNA and proteins of the three hCyPs in differing tissues and cell types was similar. Each hCyP protein was expressed in Escherichia coli, purified, and shown to be an active peptidyl-prolyl isomerase. Substrate specificity was examined with 11 synthetic peptides (Suc-Xaa-Yaa-Pro-Phe-4-nitroanilide), and inhibition of the peptidyl-prolyl isomerase activities associated with hCyP1, hCyP2, and hCyP3 was studied with CsA, MeAla6-CsA and MeBm2t1-CsA. From both equilibrium considerations and the results of kinetic characterizations it is proposed that of these three CyP proteins, hCyP1 is the most likely intracellular target for CsA.

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

A Candida albicans cDNA and its genomic counterpart were isolated from λ phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 ( c y clo p hilin ) . Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.

We have expressed fragments of the cDNA coding for mature human interleukin-1 alpha (hIL-1 alpha) in Saccharomyces cerevisiae. Mature hIL-1 alpha contains one potential N-linked glycosylation site that is not recognized in mammalian cells. Translational fusions to either one of three yeast signal sequences resulted in secretion of bioactive, N-glycosylated hIL-1 alpha. The extent of glycosylation was significantly reduced using the alpha-factor signal sequence, which itself contains three N-linked glycosylation sites known to be core glycosylated. N-glycosylation has no effect on biological specific activity.

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by the trans-acting products of the four MAR/SIR loci. MAR/SIR gene mutations result in the simultaneous derepression of HML and HMR gene expression. The sum1-1 mutation was previously identified as an extragenic suppressor of mutations in MAR1 (SIR2) and MAR2 (SIR3). As assayed genetically, sum1-1 is capable of restoring repression of silent mating-type information in cells containing mar1 or mar2 null mutations. We show here that the mating-type phenotype associated with sum1-1 results from a dramatic reduction in the steady-state level of HML and HMR gene transcripts. At the same time, the sum1-1 mutation has no significant effect on the level of each of the four MAR/SIR mRNAs.

The gastrointestinal tract microbiome has been suggested as a potential therapeutic target for metabolic diseases such as obesity and Type 2 diabetes mellitus (T2DM). However, the relationship between changes in microbial communities and metabolic disease-phenotypes are still poorly understood. In this study, we used antibiotics with markedly different antibacterial spectra to modulate the gut microbiome in a diet-induced obesity mouse model and then measured relevant biochemical, hormonal and phenotypic biomarkers of obesity and T2DM. Mice fed a high-fat diet were treated with either ceftazidime (a primarily anti-Gram negative bacteria antibiotic) or vancomycin (mainly anti-Gram positive bacteria activity) in an escalating three-dose regimen. We also dosed animals with a well-known prebiotic weight-loss supplement, 10% oligofructose saccharide (10% OFS). Vancomycin treated mice showed little weight change and no improvement in glycemic control while ceftazidime and 10% OFS treatments induced significant weight loss. However, only ceftazidime showed significant, dose dependent improvement in key metabolic variables including glucose, insulin, protein tyrosine tyrosine (PYY) and glucagon-like peptide-1 (GLP-1). Subsequently, we confirmed the positive hyperglycemic control effects of ceftazidime in the Zucker diabetic fatty (ZDF) rat model. Metagenomic DNA sequencing of bacterial 16S rRNA gene regions V1-V3 showed that the microbiomes of ceftazidime dosed mice and rats were enriched for the phylum Firmicutes while 10% OFS treated mice had a greater abundance of Bacteroidetes. We show that specific changes in microbial community composition are associated with obesity and glycemic control phenotypes. More broadly, our study suggests that in vivo modulation of the microbiome warrants further investigation as a potential therapeutic strategy for metabolic diseases.

Members of the death receptor family play a prominent role in developmental and pathological neuronal cell death. The death signal is transduced via interaction between the death domain of the receptor and an intracellular adapter, TRADD. We performed alanine-scanning mutagenesis of specific charged residues in the TR3 death domain to determine whether they play a crucial role in TR3-TR3 and TR3-TRADD interaction. Mutation of charged residues in the second and third helices of the TR3 death domain failed to perturb self-interaction or interaction with TRADD. These data suggest that despite some similarity between the death domains of TR3 and TNFR1 the nature of the interaction with TRADD differs from that reported for TNFR1.

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by cotransforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).

Uridine 5′-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

Rapamycin (Rm) and FK506 are macrolide antifungal agents that exhibit potent immunosuppressive properties in higher eukaryotes which are mediated through interaction with specific receptor proteins (FKBPs or RBps, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. We previously isolated a gene encoding an RBP from Saccharomyces cerevisiae , and demonstrated that null mutations in this gene (called RBP1 ) result in a recessive Rm-resistant (Rm R ) phentype. We now have cloned the Candida albicans RBP1 gene via complementation of the Rm R phenotype in S. cerevisiae . The predicted C. albicans RBP exhibits 61%, 52% and 49% amino acid (aa) sequence identity with RBPs (FKBPs) from S. cerevisiae, Neurospora crassa and human cells (FKBP-12), respectively. Furthermore, several of the aa residues identified as being important for drug binding in human FKBP-12 are conserved within the C. albicans RBP.

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.

To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and 3.9 +/- 0.9 and 0.5 +/- 0.1 nM, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201-886. In contrast, the IC50 values for cAMP competition with either [3H]-(R)-rolipram or [3H]RP 73401 binding increased approximately 2-fold in H505N and approximately 13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in the K(m) value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4-selective inhibitors.

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.

The primary step in the aromatic amino-acid biosynthetic pathway in Saccharomyces cerevisiae is catalyzed by two redundant isozymes of 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase, either of which alone is sufficient to permit growth on synthetic complete media lacking aromatic acids (SC-Aro). The activity of one isozyme (encoded by the ARO3 gene) is feedback-inhibited by phenylalanine, whereas the activity of the other isozyme (encoded by the ARO4 gene) is feedback-inhibited by tyrosine. Transcription of both genes is controlled by GCN4. We previously cloned the ARO3 gene from the opportunistic pathogen Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine; and (2) its expression is induced in response to amino-acid deprivation, consistent with the presence of two putative GCN4-responsive promoter elements (Pereira and Livi 1993, 1995). To determine whether other DAHP synthases exist in C. albicans, we have constructed a homozygous aro3-deletion mutant strain. Such a mutant was found to be phenotypically Aro+, i. e., capable of normal growth on SC-Aro media, suggesting the presence of at least one additional isozyme. To confirm this result, a 222-bp DNA fragment was amplified by the polymerase chain reaction (PCR) from genomic DNA prepared from the homozygous aro3-deletion mutant, using a degenerate primer based on a conserved N-terminal region of Aro3p plus a degenerate comeback primer encoding a conserved region of the protein that lies within the deleted portion of the gene. The nucleotide sequence of this PCR fragment predicts a 74-amino acid DAHP synthase-related protein which shows strong homology to Aro3p from S. cerevisiae and C. albicans, but even greater homology (78% identity) to S. cerevisiae Aro4p. We conclude that cells of C. albicans contain a second Aro4p-related DAHP synthase.

The yeast TOR1 (DRR1) and TOR2 (DRR2) proteins are putative targets of the immunosuppressive drug rapamycin (Rm), defined by dominant drug-resistance mutations. They share a large C-terminal domain that exhibits sequence similarity to the 110-kDa subunit of phosphatidylinositol (PI) 3-kinases. In this report, we present an amino acid (aa) sequence alignment of TOR1 (DRR1) and TOR2 (DRR2) and identify conserved and nonconserved motifs within the N-terminal domain that are indicative of possible nuclear localization. We also show that the mutations responsible for Rm resistance in four independent drr2dom alleles alter the identical aa (Ser1975-->Arg) previously identified in drr1dom mutants (Ser1972-->Arg or Asn). Models for TOR (DRR) protein function are discussed.

In Saccharomyces cerevisiae , the primary step in the aromatic (ARO) amino acid (aa) biosynthetic pathway is catalyzed by two isozymes of 3-deoxy- d -arabinoheptulosonate-7-phosphate synthase (DAHPS). The activity of one DAHPS isozyme (encoded by the ARO3 gene) is feedback inhibited by phenylalanine, whereas the other (encoded by the ARO4 gene) is inhibited by tyrosine. The expression of these genes is also regulated at the transcriptional level by the general control activator GCN4. We took advantage of the high degree of aa sequence homology between DAHPSs from several species to isolate ARO3 homologues from the pathogenic yeast Candida albicans . An ARO3/ARO4-specific sequence was generated from C. albicans genomic DNA by polymerase chain reaction amplification and used as a probe to screen a C. albicans cDNA library. A 1.3-kb cDNA clone was isolated and sequenced. The cDNA contains a long open reading frame predicting a 368-aa protein with significant homology to known DAHPSs, including both the S. cerevisiae ARO3 and ARO4 products (68.5% and 58.5% identity, respectively). Northern analysis of yeast and mycelial poly(A) + RNA revealed equivalent expression of a 1.3-kb transcript in both cell types. A genomic clone was isolated by crosshybridization, and analysis of the 5' untranslated region revealed the presence of a putative GCN4-binding site. This clone complemented an aro3 mutation in S. cerevisiae ; functional complementation was inhibited by the presence of excess phenylalanine (but not tyrosine) in the growth medium, confirming that the cloned gene is the C. albicans homologue of ARO3 .

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent Rmrrbp1 mutants and screened them for both RB1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm. Analysis of [3H]Rm binding in rbp1 mutant vs. wild-type yeast cell extracts indicated that the Tyr89 → Asp (Y89D) change results in an approx. 95% loss of binding activity. These data confirm the assignment of this Tyr residue as being involved in Rm binding.