The E3 ubiquitin ligase WWP1 sustains the growth of acute myeloid leukaemia.

The E3 ubiquitin ligase (E3) WWP1 is an oncogenic factor implicated in the maintenance of different types of epithelial cancers. The role of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in haematological neoplasms remains unknown. Acute myeloid leukaemia (AML) is characterized by the expansion of malignant myeloid cells blocked at different stages of differentiation. Here we report that the expression of WWP1 is significantly augmented in a large cohort of primary AML patients and in AML cell lines, compared with haematopoietic cells from healthy donors. We show that WWP1 inactivation severely impairs the growth of primary AML blasts and cell lines in vitro. In vivo, we observed a reduced leukaemogenic potential of WWP1-depleted AML cells upon transplantation into immunocompromised mice. Mechanistically, WWP1 inactivation induces the accumulation of its protein substrate p27Kip1, which ultimately contributes to G0/G1 cell cycle arrest of AML blasts. In addition, WWP1 depletion triggers the autophagy signalling and reduces survival of leukaemic cells. Collectively, our findings provide molecular insights into the anti-cancer potential of WWP1 inhibition, suggesting that this E3 is a promising biomarker and druggable target in AML.

A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae.

Gene tagging can be achieved by homologous recombination in yeast. The kan(r) marker gene plays an important role in PCR-mediated gene disruption and PCR-mediated epitope tagging experiments. In this paper, new modules containing a tag-loxP-kanMX-loxP cassette are described that allow tagging of different genes by using the kan(r) marker repeatedly.

Evidence for overlapping and distinct functions in protein transport of coat protein Sec24p family members.

Budding of transport vesicles from the endoplasmic reticulum in yeast requires the formation, at the budding site, of a coat protein complex (COPII) that consists of two heterodimeric subcomplexes (Sec23p/Sec24p and Sec13p/Sec31p) and the Sar1 GTPase. Sec24p is an essential protein and involved in cargo selection. In addition to Sec24p, the yeast Saccharomyces cerevisiae expresses two non-essential Sec24p-related proteins, termed Sfb2p (product of YNL049c) and Sfb3p/Lst1p (product of YHR098c). We here show that Sfb2p and, less efficiently, Sfb3p/Lst1p are able to bind, like Sec24p, the integral membrane cargo protein Sed5p. We also demonstrate that Sfb2p, like Sec24p and Sfb3p/Lst1p, forms a complex with Sec23p in vivo. Whereas the deletion of SFB2 did not affect transport kinetics of various proteins, the maturation of the glycolipid-anchored plasma membrane protein Gas1p was differentially impaired in sfb3 knock-out cells. We generated several conditional-lethal sec24 mutants that, combined with null alleles of SFB2 and SFB3/LST1, led to a complete block of transport between the endoplasmic reticulum and the Golgi (sec24-11/Deltasfb2) or to cell death (sec24-11/Deltasfb3). Of the Sec24p family members, Sfb2p is the least abundant at steady state, but high intracellular concentrations of Sfb2p can rescue sec24 mutants under restrictive conditions. The data presented strongly suggest that the Sec24p-related proteins function as COPII components.

Specific interaction of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II component Sec24p of endoplasmic reticulum-derived transport vesicles.

The generation of transport vesicles at the endoplasmic reticulum (ER) depends on cytosolic proteins, which, in the form of subcomplexes (Sec23p/Sec24p; Sec13p/Sec31p) are recruited to the ER membrane by GTP-bound Sar1p and form the coat protein complex II (COPII). Using affinity chromatography and two-hybrid analyses, we found that the essential COPII component Sec24p, but not Sec23p, binds to the cis-Golgi syntaxin Sed5p. Sec24p/Sed5p interaction in vitro was not dependent on the presence of [Sar1p.GTP]. The binding of Sec24p to Sed5p is specific; none of the other seven yeast syntaxins bound to this COPII component. Whereas the interaction site of Sec23p is within the N-terminal half of the 926-aa-long Sec24p (amino acid residues 56-549), Sed5p binds to the N- and C-terminal halves of the protein. Destruction by mutagenesis of a potential zinc finger within the N-terminal half of Sec24p led to a nonfunctional protein that was still able to bind Sec23p and Sed5p. Sec24p/Sed5p binding might be relevant for cargo selection during transport-vesicle formation and/or for vesicle targeting to the cis-Golgi.

Combination of a drug targeting the cell with a drug targeting the virus controls human immunodeficiency virus type 1 resistance.

Combinations of drugs targeting viral proteins have been used to limit or control drug resistance, which is the most important cause of treatment failure in HIV-1-infected individuals. We suggest an alternative approach, namely to target cellular proteins, which are less prone to mutations than viral proteins. Here we show that simultaneous inhibition of a cellular protein (by hydroxyurea) and a viral protein (by ddI) produces a consistent and sustained suppression of HIV-1 for as long as 40 weeks in the absence of virus rebound. We identified the mechanism to explain this lack of rebound: although the combination of the two drugs did not prevent the emergence of mutant viral strains resistant to didanosine (ddI) in these patients, the mutants were still sensitive to standard doses of ddI in the presence of hydroxyurea. These in vivo results were consistent with our in vitro observations: HIV-1 molecular clones resistant to ddI were rendered sensitive to this drug (at concentrations routinely achievable in vivo) after addition of hydroxyurea. This phenomenon can be explained by the observation that hydroxyurea decreases the level of dATP, the cellular competitor of ddI. A low level of dATP favors the incorporation of ddI, even if the viral reverse transcriptase is resistant to this nucleoside analog. This is a novel mechanism of control of resistance and it explains the efficacy of a treatment that is well tolerated, simple, and inexpensive.

Telethonin, a novel sarcomeric protein of heart and skeletal muscle.

In this paper we describe a novel 19 kDa sarcomeric protein named telethonin. The cDNA sequence discloses an open reading frame of 167 amino acids that does not resemble any known protein. Antibodies against a recombinant telethonin fragment were used for Western blot analysis, confirming the presence of this 19 kDa protein in heart and skeletal muscle and revealing an immunofluorescence pattern typical of sarcomeric proteins, overlapping myosin. The frequency of specific cDNA clones in different libraries indicates that the telethonin transcript is amongst the most abundant in skeletal muscle. In human, telethonin maps at 17q12, adjacent to the phenylethanolamine N-methyltransferase gene.

Mutations in the pol gene of human immunodeficiency virus type 1 in infected patients receiving didanosine and hydroxyurea combination therapy.

The pattern of mutations in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) strains that confer resistance to didanosine (ddI) was analyzed in 2 groups of patients receiving either ddI monotherapy or ddI plus hydroxyurea (HU) combination therapy. Twelve patients receiving combination therapy and 8 receiving monotherapy were tested. Combinations of ddI plus HU did not prevent the onset of mutations, which emerged in 50% of the patients in this group compared with 25% of the ddI monotherapy group. In addition, in 1 patient from the combination therapy arm, who had a limited response to the therapy, an unusual pattern of mutations was found: the insertion of 2 amino acids between residues 69 and 70, a region critical for resistance to nucleoside analogs. The higher efficacy of the combination of HU and ddI compared with that of ddI monotherapy cannot be attributed to a delayed or decreased onset of resistance to ddI.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

The DNA sequence of cosmid 14-13b from chromosome XIV of Saccharomyces cerevisiae reveals an unusually high number of overlapping open reading frames.

This work is part of the effort for sequencing chromosome XIV of Saccharomyces cerevisiae. Cosmid 14-13b contains a 37.8 kb insert derived from a partial Sau3A digestion of the genome, cloned into the BamHI site of the vector Pou6. The strategy used for sequencing is based on the fragmentation of the whole cosmid by sonication, followed by shotgun sequencing on an Applied Biosystem DNA sequencer. The clones with inserts corresponding to the vector were identified by dot-blot hybridization, without the need of sequencing. The analysis of the DNA sequence reveals 29 open reading frames (ORFs) longer than 300 bases. Nine ORFs are internal to some other ORFs. Similarity searches against DNA and protein data banks show that six ORFs correspond to already known yeast genes (OMP1, PSU1, MLS1, RPC19, DBP2, CYB5) and one ORF matches the sequence of a putative yeast gene (ESBP6).

The DNA sequence of cosmid 14-5 from chromosome XIV reveals 21 open reading frames including a novel gene encoding a globin-like domain.

In this paper is described the DNA sequence of cosmid 14-5 from chromosome XIV of Saccharomyces cerevisiae. The sequence is 38 855 bases long and contains 21 open reading frames (ORFs) plus three internal ORFs. Six ORFs correspond to known yeast genes (SLA2, ZWF1, BLH1, KEX2, SIN4 and URE2); two other ORFs had already been sequenced because they are adjacent to known genes; the remaining 12 ORFs are novel genes. Of these, one ORF (NII42) is particularly interesting since it shows a significant similarity to mammalian globin. Another ORF (N1254) displays two zinc finger motifs as well as a DNAJ motif.

Metabolites and enzyme activities involved in tryptophan metabolism in two different strains of mouse.

The urinary excretion of the tryptophan metabolites along the kynurenine pathway and the variations of enzyme activities involved in the degradation of tryptophan have been studied in two strains of mice. The total excretion of the metabolites was significantly higher in the strain of albino N.C.L. than in Swiss albino mice, in accordance with the higher activity of tryptophan pyrrolase, which was present only as holoenzyme. In both strains kynurenine, kynurenic and xanthurenic acids were excreted in larger amounts. However, the albino N.C.L. mice excreted a large amount of xanthurenic acid, not correlated with the slightly higher kynurenine aminotransferase activity observed in this strain. The very high excretion of this metabolite indicates that the load of tryptophan causes a B6-deficiency. Liver kynureninase activity was similar in both strains. Correlation between total urinary excretion of the tryptophan metabolites and enzyme activities appears in the same strain of mice, even though differences are present in different strains.

Enzyme activities and metabolites along the kynurenine pathway in mice with Harding-Passey melanoma.

Tryptophan metabolism has been studied in mice with Harding-Passey melanoma and in controls, after a load of 1.0 g/kg b.w. of L-tryptophan by determining ten urinary metabolites of the kynurenine pathway and some enzyme activities involved in the degradation of this aminoacid. Kynurenine was the only tryptophan derivative excreted in significantly higher quantities in mice with melanoma with respect to the controls. This result is in agreement with a significantly higher activity of hepatic tryptophan pyrrolase in mice with melanoma. Liver kynureninase and liver and kidney kynurenine aminotransferase activities were similar in the two groups of mice. These findings point to a possible role of tryptophan in the biogenesis of melanins in pathological conditions such as in melanoma.

Strain differences in the tryptophan metabolite excretion and enzyme activities along the kynurenine pathway in rats.

The strain differences of the urinary excretion of the tryptophan metabolites along the kynurenine pathway and the variations of enzyme activities metabolizing the tryptophan have been studied in different strains of rats. There is a good correlation between urinary excretory values of the metabolites and enzyme activities in the same strain of rats. However, some differences appear when the data are compared among different strains. Wistar, heterozygous Gunn and Sprague-Dawley rats show similar total urinary excretion of tryptophan metabolites, while the Long Evans rats have significantly lower values, in accordance with the lower activity of tryptophan pyrrolase. Kynureninase activity is slightly but not significantly higher in Sprague-Dawley rats, in agreement with the high levels of anthranilic acid excreted. As regards kynurenine aminotransferase, Sprague-Dawley rats show lower activity in the liver, but higher activity in the kidneys in comparison to other strains of Wistar, Long Evans and heterozygous Gunn rats. The importance of considering strain differences is emphasized when making comparisons of measurements carried out in different laboratories.

Alterations of the tryptophan--nicotinic acid metabolism in vitiligo.

Tryptophan metabolism "via kynurenine" is altered in vitiligo: after a load of the amino acid the urinary excretion of 3-hydroxykynurenine and 3-hydroxyanthranilic acid is decreased, whereas that of xanthurenic acid and its 8-methyl ether is increased. The excretory values of the metabolites suggest a deficiency of the activity of kynurenine hydroxylase and kynunerinase, the enzymes involved in the metabolism of 3-hydroxykynurenine and 3-hydroxyanthranilic acid. The reduced excretion of 3-hydroxykynurenine, a tryptophan metabolite involved in melanin biosynthesis, may indicate a smaller utilization of tryptophan in the biogenesis of the melanins.

[Tryptophan metabolism in patients with vitiligo]. / Studi sul metabolismo del triptofano in pazienti con vitiligine.

Our previous research showed that tryptophan is an important precursor in the biogenesis of melanins. Therefore, with the purpose of observing whether a relationship exists between tryptophan metabolism and diseases characterized by an altered process of skin pigmentation in man, we studied the metabolism of this aminoacid along the kynurenine pathway in 29 vitiliginous patients (11 males and 18 females) and in 21 control subjects (11 males and 10 females) by determining 10 urinary metabolites after an oral loading of 50 mg/kg body weight L-tryptophan. The mean total excretion of the metabolites in patients resulted similar to that of the controls. However, considering the individual metabolites one can observe a decreased excretion of 3-hydroxykynurenine, o-aminohippuric acid and 3-hydroxyanthranilic acid and an increased excretion of xanthurenic acid and of its 8-methyl ether in the group of vitiliginous patients in respect to the controls. These results seem to indicate a decreased formation of nicotinic acid from tryptophan. Moreover, in relation to the fact that 3-hydroxykynurenine could be the metabolite through which tryptophan is involved in melanin biosynthesis, this study supports the hypothesis of a connection of tryptophan metabolism with the lack of pigmentation in vitiligo.

Effect of blue light on the urinary excretion of tryptophan metabolites in adult heterozygous and homozygous Gunn rats.

Tryptophan metabolism along the kynurenine pathway has been studied in male and female heterozygous and homozygous adult Gunn rats after load of amino acid before and after exposure to blue light. The percentage of tryptophan metabolites in each group of rats was slightly higher in females than in males. Before irradiation the excretion of metabolites was larger in male and female heterozygous groups than the corresponding groups of the homozygotes. After irradiation only the groups of male and female heterozygous Gunn rats showed a decrease in the total excretion of the metabolites. Therefore blue light exposure dose not seem to influence the metabolism of tryptophan in homozygous Gunn rats.

Enzyme activities of tryptophan metabolism in heterozygous and homozygous Gunn rats before and after blue light exposure.

The effect of light exposure in the enzyme activities, involved in the tryptophan metabolism along the kynurenine pathway, has been studied in male and female adult hetero-and homozygous Gunn rats. Light exposure did not seem to influence these enzyme activities. The groups of non-icteric heterozygotes however showed a higher activity of liver tryptophan pyrrolase than in the groups of icteric homozygotes. Kynureninase activity was similar in all the groups of rats, whereas liver kynurenine aminotransferase activity appeared slightly higher in the groups of male rats with respect to female rats, but no difference existed before and after irradiation. In addition the male heterozygotes showed a higher kynurenine aminotransferase activity in kidneys than the other groups of rats.