Pascual Sanz

Carbon Source-Dependent Phosphorylation of Hexokinase PII and Its Role in the Glucose-Signaling Response in Yeast

ABSTRACT The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism inSaccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PII is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, andcid1/glc7. Treatment of a glucose crude extract fromcid1-226 (glc7-T152K) mutant cells with λ-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2(S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction ofHXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.

Stable High-Copy-Number Integration of Aspergillus oryzae α-AMYLASE cDNA in an Industrial Baker's Yeast Strain

The Aspergillus oryzae α‐amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial bakers yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the α‐amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High‐copy‐number integrants were obtained including a defective TRP1d promoter in the integrative cassette. We selected one transformant, Rib‐AMY (CECT10872), in which the multi‐integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of α‐amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib‐AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous α‐amylase to the flour, reducing allergenic work‐related symptoms due to this enzyme.

Cell Wall Proteins Liberated by Zymolyase from Several Ascomycetous and Imperfect Yeasts

SUMMARY: Eight yeast species (the ascomycetes Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Hansenula wingei, Saccharomycopsis lipolytica, Schizosaccharomyces pombe and Pichia scolyti, and the imperfect yeasts Candida albicans and Rhodotorula glutinis) have been compared with respect to the proteins solubilized by glucanase treatment of amino-acid-labelled, purified walls. Except for R. glutinis, a significant quantity of radioactively labelled protein material was liberated by this treatment. Among the major protein components solubilized was a material larger than 100 kDa (heterogeneous in size in some species) and a molecule ranging in size from 31.5 to 34 kDa depending on the yeast species. The large material was of glycoprotein nature, with the sugar portion N-glycosidically linked to the protein moiety; in some species, this material did not bind concanavalin A (ConA), indicating the presence of terminal sugar residues different from mannose, glucose or glucosamine. The form of 31.5-34 kDa was present in all the species studied except R. glutinis; it was a mannoprotein, except in Schiz. pombe, which possessed a 31.5 kDa form not sensitive to tunicamycin or endoglycosidase H and not recognized by ConA. Antigenic cross-reactivity was observed between the protein moieties of the 33 kDa form of Sacch. cerevisiae and the species of equivalent size in C. albicans and H. wingei. Similar partial proteolysis patterns were obtained for the 33 kDa form of Sacch. cerevisiae and the 34 kDa forms of C. albicans and P. scolyti.

Secretory pattern of a major integral mannoprotein of the yeast cell wall

Abstract A major integral mannoprotein of the Saccharomyces cerevisiae cell wall with an apparent size of 33 kDa has been purified and polyclonal antibodies have been raised against its enzymatically deglycosylated protein moiety. These have been employed to immunodetect the molecular forms of this species accumulated in yeast sec mutants. Microsomal preparations from those mutants blocked at the initial steps of the secretory route ( sec53 and sec59 ) contained the 30.5 kDa non-glycosylated form, although minor amounts of partially glycosylated forms and the 33 kDa one were also immunodetected in sec53 membranes. Mutants blocked at later steps ( sec18 , sec7 and sec1 ) accumulated only the glyciosylated form. Criteria such as sensitivity to pronase and solubilization by Triton-urea in mild conditions have been employed to study the topology of the 30.5 and 33 kDa forms in intact microsomal vesicles. In sec53 and sec59 cells, the non-glycosylated form accumulates at the cytoplasmic face of the endoplasmic reticulum, although in the case of the sec59 mutant the molecules appear to be tightly stuck to the membrane. In sec1 and sec18 cells, the 33 kDa mannoprotein is apparently localized at the vesicular lumen as a peripheral-membrane form or as an entirely soluble one.

The Bacillus subtilis lipoprotein LplA causes cell lysis when expressed in Escherichia coli

A gene called lplA (lipoprotein-like) has been isolated from a genomic library of Bacillus subtilis expressed in Escherichia coli. Clones carrying the lplA gene were selected by the ability of the colonies to give visible haloes of starch hydrolysis. The cloned fragment contains an open reading frame (ORF) of 1509 bp encoding a protein of 56 kDa. The protein contains a typical N-terminal signal sequence, a putative transmembrane anchor domain and a leucine zipper at the C-terminus. The expression of this protein in E. coli causes cell lysis, only the N-terminal domain of the LplA protein being responsible for this phenotype. The mechanism of cell lysis is similar to that previously suggested for the expression in E. coli of the lipoproteins encoded by the Streptococcus pneumoniae genes malX and amiA. The protein is modified with palmitic acid when secreted in E. coli, confirming that it is a typical lipoprotein.

Characterization of novel neopullulanase fromBacillus polymyxa

Bacillus polymyxa CECT 155 produces an extracellular neopullulanase activity that degrades pullulan to panose. This activity was stimulated by the presence of pullulan in the culture, and repressed by glucose. The apparent mol wt determined for the enzyme was 58 kDa. The optimum pH and temperature for neopullulanase activity were pH 6.0 and 50°C, respectively. The enzyme was stable in a pH range of 4.0–8.0, and temperatures up to 60°C. These properties make it suitable for the saccharification processes in the starch industries.

Autolytic Release of Mannoproteins from Cell Walls of Saccharomyces cerevisiae

Summary: Autolysis of purified Saccharomyces cerevisiae cell walls resulted in the release of several components thought to be mannoprotein in nature, since they were retained by Concanavalin A-Sepharose. The most abundant was a 29 kDal molecule which was also a major species among mannoproteins solubilized by the β-glucanase complex Zymolyase. There was a concomitant release of glucose and mannose oligosaccharides and of β-glucanase activity. Glucanase and protease inhibitory treatments considerably lowered the release of mannoproteins. The use of different protease inhibitors during autolytic incubations interfered with at least two proteases (one of them being a cysteine protease) apparently involved in the solubilization and partial degradation of mannoproteins from the walls.

Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans.

Introduction Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest. Material and Methods A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated. Results Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p<0.001) than the reference standards in all cases. Conclusion Hydrofection of hAAT DNA to “in vivo” isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein expression. Impairment of protein secretion to plasma is observed and might be species-related. This study reinforces the potential application of closed liver hydrofection for therapeutic purposes, provided protein secretion improves.

Energy consumption as a predictor test of the durability of a biological tissue employed in cardiac bioprosthesis

The mechanical behavior of the young bull pericardium in a fatigue test has been studied. This material is a similar tissue to those used in valve leaflet construction for a cardiac bioprosthesis. The consumed energy on each test was evaluated and afterwards used as a predictor of the biomaterial strength. Two-hundred and nine samples were tested to cyclical fatigue. The cut-off point to determine the sample quality was whether or not they resisted at least 4500 cycles. Only 22 samples withstood over that point (10.52%). The samples were classified according to their fatigue behavior in excellent, undefined and unsuitable. By using as a reference the consumed energy in the first 25 cycles, we could distinguish correctly (between 93.2 and 96.1%) the unsuitable material and most of the excellent (between 78.1 and 95.2%). From the rejected material 77% was really detachable and from the accepted, only 50% was excellent, with an equal methodology. The receiver operating characteristics curve was employed to establish decision levels when selecting samples, being 0.85 the best area (theoretical maximum value of 1). It is concluded that the energy wasted is a good predictor of the strength of the tissue. More than 90% of the unsuitable material and 50% of the excellent material (5% of all the material) is detected with this method.

Lafora Disease: A Ubiquitination-Related Pathology

Lafora disease (LD, OMIM254780) is a rare and fatal form of progressive myoclonus epilepsy (PME). Among PMEs, LD is unique because of the rapid neurological deterioration of the patients and the appearance in brain and peripheral tissues of insoluble glycogen-like (polyglucosan) inclusions, named Lafora bodies (LBs). LD is caused by mutations in the EPM2A gene, encoding the dual phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Laforin and malin form a functional complex that is involved in the regulation of glycogen synthesis. Thus, in the absence of a functional complex glycogen accumulates in LBs. In addition, it has been suggested that the laforin-malin complex participates in alternative physiological pathways, such as intracellular protein degradation, oxidative stress, and the endoplasmic reticulum unfolded protein response. In this work we review the possible cellular functions of laforin and malin with a special focus on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD.