Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.     

Optimization of mycophenolic acid production in solid state fermentation using response surface methodology

Mycophenolic acid (MPA) can be produced in solid state fermentation. An isolate of Penicillium brevi-compactum ATCC 16024 grown on moist wheat bran produced a titre of 425 mg per kg of wheat bran. Central composite rotatable design and response surface methodology were employed to derive a statistical model for media optimization towards production of mycophenolic acid. Five levels with a five factorial design were adopted. The correlation coefficient was 0.82, ensuring a satisfactory adjustment of the model to the experimental values. This statistical design was very effective in improving the titre of mycophenolic acid up to 3286 mg per kg of wheat bran. Received 24 July 1998/ Accepted in revised form 4 December 1998     

Transformation of the yeast Hansenula polymorpha by a hybrid plasmid containing the fragment of host DNA

Spheroplasts of Hansenula polymorpha strain deficient in 2-isopropylmalate dehydrogenase have been shown to be transformed by the DNA of a hybrid plasmid pHRI, carrying the LEU2 gene from S. cerevisiae and 2.0 kilobase HindIII fragment of H. polymorpha genomic DNA. The frequency of transformants has reached 10(3) per 1 microgram of transforming DNA. Plasmid pHRI is maintained in transformants as an autonomous circular DNA molecule and is inherited by 1-2% fraction of cells from the population growing under the selective conditions. Transformation takes place under the same conditions that are required for spheroplast fusion. Thus, H. polymorpha becomes one more species of yeast susceptible to hybrid plasmid-mediated gene transfer in the process of DNA transformation.     

Role of the hypothalamus in originating disorders of exploratory behavior and learning in rats with excised nuclei of the locus coeruleus

Open-field behaviour and emotionally differently reinforced learning were studied in male Wistar rats with bilaterally ablated Locus coeruleus. Histochemical analysis of the hypothalamic structures was carried out. Decrease of investigating activity and attention was found as well as disturbances of learning with emotionally-negative (painful) reinforcement. By means of histochemical methods, fluorescence characteristic for catecholamines was found to decrease sharply in paraventricular and supraoptic hypothalamic nuclei, eminentia medialis and the posterior lobe of the hypophysis.     

Structural heterochromatin and X-chromosome inactivation]

Our previous studies on the expression of the G6PD and alpha-GAL genes from the X chromosome of inter-specific hybrids of voles of the Microtus genus have demonstrated an unusual pattern of X-inactivation in the parents. The observed phenomenon was explained as the presumable result of nonrandom inactivation of the X chromosomes with a heterochromatin block in crosses involving Microtus arvalis whose X lacks a heterochromatin region and also of random X inactivation when both parents had heterochromatin blocks on the Xs. Based on known models, we discuss here the possible mechanisms of the effect of heterochromatin on X-inactivation; we give preference to the model postulating binding of nonhistone protein to the inactivation centre as the key event. The hypothesis we offer suggests change in chromatin conformation in the inactivation centre during packaging of heterochromatic region of a chromosome; the protein molecules diffusing along the chromosome towards the heterochromatin region by the "facilitated diffusion" mechanism may happen to be in the region of the X-inactivation centre, which, being in a favorable state, binds specifically to it; as a consequence, the binding probability of protein to heterochromatin increases as compared to chromosome without heterochromatin block.     

Properties of proteolytic enzymes isolated from a thermophilic strain of Micromonospora vulgaris 42.

Two proteolytic enzymes, protease A and protease B, were isolated in homogeneous state from the cultural broth of the thermophilic actinomycete Micromonospora vulgaris 42. Their physicochemical properties were studied, i.e., molecular weight (50 000 for protease A and 30 000 for protease B), amino acid composition, N-terminal amino acids (phenylalanine for protease A and alanine for protease B). The specificity of the action of these enzymes was assayed by splitting the B chain of oxidized insulin. Both enzymes are neutral proteases of the thermolysine type.     

Effect of the regulatory subunit of cAMP-dependent protein kinase on the genetic activity of eukaryotic cells

Summary The effect of the regulatory subunit of cAMP-dependent protein kinase from pig brain on the protein synthesis in normal (3T3) and virus-transformed (SV40-3T3) cells was studied. The regulatory subunit was found to induce a specific synthesis of new proteins; the direct and first response of 3T3 cells to the introduction of the regulatory subunit being the synthesis of the protein P-15. The molecular weight of the protein was 15 000, the isoelectric point 6.3. The electrophoretic analysis of the cytosol of SV40-3T3 cells demonstrated a general derepression of the genome of the virus-transformed cells. A protein identical with P-15 was detected to be present in SV40-3T3 cells. The treatment of these cells with the regulatory subunit as well as with cAM P separately did not affect the synthesis of P-15, whereas the introduction of the cAM P-regulatory subunit complex caused a significant expression of the protein P-15. The data obtained indicate that the protein synthesis is dependent on the nuclear translocation of the regulatory subunit.     

Virus-like particles in yeast: isolation and infectivity.

Virus-like particles containing electron dense cores are seen in thin sections of intact and degenerated cells of a thermosensitive (ts) strain of Candida tropicalis. A particulate fraction not present in wild-type cells has been isolated from the ts cells disrupted by pressure. The particles are 80-120 nm in diameter. Empty particles with a central cavity are observed. The method of infecting mating pairs of Saccharomyces cerevisiae by partially purified particles is described.     

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.