Jun Urano

Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-Like proteins

Early in development, a part of the embryo is set aside to become the germ cell lineage that will ultimately differentiate to form sperm and eggs and transmit genetic information to the next generation. Men with deletions encompassing the Y-chromosome DAZ genes have few or no germ cells but are otherwise healthy, indicating they harbor specific defects in formation or maintenance of germ cells. A DAZ homolog, DAZL (DAZ-Like), is found in diverse organisms, including humans and is required for germ cell development in males and/or females. We identified proteins that interact with DAZ proteins to better understand their function in human germ cells. Here, we show that PUM2, a human homolog of Pumilio, a protein required to maintain germ line stem cells in Drosophila and Caenorhabditis elegans, forms a stable complex with DAZ through the same functional domain required for RNA binding, protein–protein interactions and rescue of Pumilio mutations in flies. We also show that PUM2 is expressed predominantly in human embryonic stem cells and germ cells and colocalizes with DAZ and DAZL in germ cells. These data implicate PUM2 as a component of conserved cellular machinery that may be required for germ cell development.

Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy

Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3–5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50–60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5–10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells

Rheb is a unique member of the Ras superfamily GTP-binding proteins. We as well as others previously have shown that Rheb is a critical component of the TSC/TOR signaling pathway. In fission yeast, Rheb is encoded by the rhb1 gene. Rhb1p is essential for growth and directly interacts with Tor2p. In this article, we report identification of 22 single amino acid changes in the Tor2 protein that enable growth in the absence of Rhb1p. These mutants also exhibit decreased mating efficiency. Interestingly, the mutations are located in the C-terminal half of the Tor2 protein, clustering mainly within the FAT and kinase domains. We noted some differences in the effect of a mutation in the FAT domain (L1310P) and in the kinase domain (E2221K) on growth and mating. Although the Tor2p mutations bypass Rhb1ps requirement for growth, they are incapable of suppressing Rhb1ps requirement for resistance to stress and toxic amino acids, pointing to multiple functions of Rhb1p. In mammalian systems, we find that mammalian target of rapamycin (mTOR) carrying analogous mutations (L1460P or E2419K), although sensitive to rapamycin, exhibits constitutive activation even when the cells are starved for nutrients. These mutations do not show significant difference in their ability to form complexes with Raptor, Rictor, or mLST8. Furthermore, we present evidence that mutant mTOR can complex with wild-type mTOR and that this heterodimer is active in nutrient-starved cells.

Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast.

Rheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine‐17, serine‐21, lysine‐120 or asparagine‐153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine‐resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP‐bound/GDP‐bound ratio of Rhb1. By using the hyperactive mutant, Rhb1K120R, we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTP‐dependent association of Rheb with Tor.

Failure to farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in the Schizosaccharomyces pombe farnesyltransferase mutant

Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology. The Schizosaccharomyces pombe mutant, cpp1–, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine. In this work, we report a novel phenotype of this mutant, enrichment of G0/G1 phase cells. This phenotype results mainly from the inability to farnesylate the Rheb G‐protein, as normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb‐CVIL) that can bypass farnesylation. In contrast, a farnesylation‐defective mutant of SpRheb (SpRheb‐SVIA) is incapable of restoring the normal cell cycle profile to the cpp1– mutant. Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle. This growth arrest phenotype of the sprheb– disruption can be complemented by the introduction of wild‐type sprheb+. The complementation is dependent on farnesylation, as the farnesylation‐defective SpRheb‐SVIA mutant is incapable of complementing the sprheb– disruption. Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb– disruption. Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb. Our findings suggest that protein farnesylation is important for cell cycle progression of S. pombe cells and that farnesylated SpRheb is critical in this process.

A Mutant Form of Human Protein Farnesyltransferase Exhibits Increased Resistance to Farnesyltransferase Inhibitors

Protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras. Recent demonstrations that inhibitors of this enzyme block the growth of a variety of human tumors point to the importance of this enzyme in human tumor formation. In this paper, we report that a mutant form of human FTase, Y361L, exhibits increased resistance to farnesyltransferase inhibitors, particularly a tricyclic compound, SCH56582, which is a competitive inhibitor of FTase with respect to the CAAX (where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) substrates. The Y361L mutant maintains FTase activity toward substrates ending with CIIS. However, the mutant also exhibits an increased affinity for peptides terminating with CIIL, a motif that is recognized by geranylgeranyltransferase I (GGTase I). The Y361L mutant also demonstrates activity with Ha-Ras and Cdc42Hs proteins, substrates of FTase and GGTase I, respectively. In addition, the Y361L mutant shows a marked sensitivity to a zinc chelator HPH-5 suggesting that the mutant has altered zinc coordination. These results demonstrate that a single amino acid change at a residue at the active site can lead to the generation of a mutant resistant to FTase inhibitors. Such a mutant may be valuable for the study of the effects of FTase inhibitors on tumor cells.

Protein Farnesylation Is Critical for Maintaining Normal Cell Morphology and Canavanine Resistance in Schizosaccharomyces pombe

Protein farnesyltransferase (FTase) plays important roles in the growth and differentiation of eukaryotic cells. In this paper, we report the identification of theSchizosaccharomyces pombe genecpp1 + encoding the β-subunit of FTase. The predicted amino acid sequence of the cpp1 + gene product shares significant similarity with FTase β-subunits from a variety of organisms. S. pombe FTase purified from E. coli exhibits high enzymatic activity toward the CAAXfarnesylation motif substrates (where C represents cysteine,A represents aliphatic amino acid, and X is preferentially methionine, cysteine, serine, alanine, or glutamine) while showing little preference for CAALgeranylgeranylation motif substrates (where L represents leucine or phenylalanine). cpp1 + is not essential for growth as shown by gene disruption; however, mutant cells exhibit rounded or irregular cell morphology. Expression of a geranylgeranylated mutant form, Ras1-CVIL, which can bypass farnesylation, rescues these morphological defects. We also identify a novel phenotype of cpp1 − mutants, hypersensitivity to canavanine. This appears to be due to a 3–4-fold increase in the rate of arginine uptake as compared with wild-type cells. Expression of the geranylgeranylated mutant form of a novel farnesylated small GTPase, SpRheb, is able to suppress the elevated arginine uptake rate. These results demonstrate that protein farnesylation is critical for maintaining normal cell morphology through Ras1 and canavanine resistance through SpRheb.

ADVANCES IN THE DEVELOPMENT OF FARNESYLTRANSFERASE INHIBITORS : SUBSTRATE RECOGNITION BY PROTEIN FARNESYLTRANSFERASE

A variety of compounds that show promise in cancer chemotherapy and chemoprevention have been identified as farnesyltransferase inhibitors. These can be classified into mainly two different types of inhibitors, farnesyl diphosphate competitors and CAAX peptidomimetics. The former type acts by competitively inhibiting farnesyltransferase with respect to one of the substrates, farnesyl diphosphate, whereas the latter type acts by mimicking the other substrate, the C‐terminal CAAX motif of Ras protein. One example of a farnesyl diphosphate competitor is manumycin, an antibiotic detected in the culture media of a Streptomyces strain. The CAAX peptidomimetics were developed based on the unique property of farnesyltransferase to recognize the CAAX motif at the C‐terminus of the protein substrate. Our recent studies have focused on understanding the structural basis of this CAAX recognition. By using in vitro mutagenesis, residues of yeast farnesyltransferase important for the recognition of the CAAX motif have been identified. Two of these residues are closely located at the C‐terminal region of the β‐subunit of farnesyltransferase. These and other results on the structural basis of the CAAX recognition may provide information valuable for structure‐based design of farnesyltransferase inhibitors. J. Cell. Biochem. Suppl. 27:12–19. Published 1998 Wiley‐Liss, Inc.

Characterization of Rheb functions using yeast and mammalian systems.

Publisher Summary This chapter presents the methods used in the study of ScRheb in the yeast, S. cerevisiae , as well as those used to study mammalian Rheb. The chapter also describes methods used to address the C-terminal farnesylation of Rheb proteins and the requirement of this modification in Rheb function. Rheb (Ras homolog enriched in brain) is a new member of the Ras superfamily of G proteins that is highly conserved in a wide range of organisms. Homologs have been identified in human, rat, Saccharomyces cerevisiae , Schizosaccharomyces pombe , fruitfly, zebrafish, sea squirt, Botrvtis cinerea , and Candida albicans . Rheb shares some of the biological properties of Rap proteins, such as binding nonproductively to Raf-I and antagonizing Ras transformation: however, Rheb also has unique attributes. Rheb shows immediate-early gene characteristics. The Rheb transcript is increased in response to maximal electroconvulsive seizures as well as to N-methyl-D-aspartate (NMDA)-mediated synaptic activity and growth factors. In addition, the protein is farnesylated and the protein is localized to the plasma membrane.

3 Mutational analyses of protein farnesyltransferase

Publisher Summary This chapter discusses deletion studies that defined the minimal size of farnesyltransferase (FTase) subunits critical for the structural integrity and function of the enzyme. The chapter describes the identification and characterization of a variety of FTase mutants that exhibit altered zinc binding, substrate affinity, and catalysis. The chapter also discusses different approaches that led to the identification of FTase mutants possessing altered substrate specificity. Some FTase mutants obtained by mutational analyses may provide valuable tools with which to evaluate the physiological function of FTase. For example, mutants resistant to farnesyltransferase inhibitor (FTI) could be used to determine whether any biological effects of FTI are indeed because of the inhibition of FTase. Both mammalian and yeast FTase mutants are discussed in the chapter, as there are striking similarities in the behavior of yeast and mammalian enzymes carrying analogous mutations.