Ribosomal stalk protein phosphorylating activities in Saccharomyces cerevisiae

With ribosomal P protein as a substrate, five peaks of protein kinase activity are eluted after chromatography of a Saccharomyces cerevisiae cellular extract on DEAE-cellulose. Two of them correspond to CK-II and the other three have been called RAP-1, RAP-II, and RAP-III. RAP-I was previously characterized. RAP-III is present in a very small amount, which hindered its purification. RAP-II was further purified on phosphocellulose, heparin-Sepharose, and P protein-Sepharose, studied in detail, and compared with other acidic protein kinases, including RAP-I, CK-II, and PK60. RAP-II is shown by SDS-PAGE and centrifugation on glycerol linear density gradients to have a molecular mass of around 62 kDa and it is immunologically different from RAP-I and PK60. RAP-II phosphorylates the P proteins in the last serine residue at the highly conserved carboxyl terminal domain as other P-protein kinases. The ribosome-bound stalk P proteins are not equally phosphorylated by the different kinases. Thus, RAP-II and PK60 mainly phosphorylate P1beta and P2alpha whereas RAP-I and CK-II modify all of them. A comparative study of the K(m) and V(max) of the phosphorylation reaction by the different kinases using individual purified acidic proteins suggests changes in the substrate susceptibility upon binding to the ribosome. All the data available reveal clear differences in the characteristics of the various P protein kinases and suggest that the cell may use them to differentially modify the stalk depending, perhaps, on metabolic requirements.     

Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms

For most neurodegenerative diseases the precise duration of an individual cell''s death is unknown, which is an obstacle when counteractive measures are being considered. To address this, we used the rd1 mouse model for retinal neurodegeneration, characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cyclic guanosine-mono-phosphate (cGMP) levels. Using cellular data on cGMP accumulation, cell death, and survival, we created mathematical models to simulate the temporal development of the degeneration. We validated model predictions using organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast. Together, photoreceptor data and modeling for the first time delineated three major cell death phases in a complex neuronal tissue: (1) initiation, taking up to 36 h, (2) execution, lasting another 40 h, and finally (3) clearance, lasting about 7 h. Surprisingly, photoreceptor neurodegeneration was noticeably slower than necrosis or apoptosis, suggesting a different mechanism of death for these neurons.     

In vivo assembling of bacterial ribosomal protein L11 into yeast ribosomes makes the particles sensitive to the prokaryotic specific antibiotic thiostrepton

Eukaryotic ribosomal stalk protein L12 and its bacterial orthologue L11 play a central role on ribosomal conformational changes during translocation. Deletion of the two genes encoding L12 in Saccharomyces cerevisiae resulted in a very slow-growth phenotype. Gene RPL12B, but not the RPL12A, cloned in centromeric plasmids fully restored control protein level and the growth rate when expressed in a L12-deprived strain. The same strain has been transformed to express Escherichia coli protein EcL11 under the control of yeast RPL12B promoter. The bacterial protein has been found in similar amounts in washed ribosomes from the transformed yeast strain and from control E. coli cells, however, EcL11 was unable to restore the defective acidic protein stalk composition caused by the absence of ScL12 in the yeast ribosome. Protein EcL11 induced a 10% increase in L12-defective cell growth rate, although the in vitro polymerizing capacity of the EcL11-containing ribosomes is restored in a higher proportion, and, moreover, the particles became partially sensitive to the prokaryotic specific antibiotic thiostrepton. Molecular dynamic simulations using modelled complexes support the correct assembly of bacterial L11 into the yeast ribosome and confirm its direct implication of its CTD in the binding of thiostrepton to ribosomes.     

Facultative response to a kleptoparasite by the cooperatively breeding pied babbler

In many cases of interspecific kleptoparasitism, hosts developdefensive behaviors to minimize the impact of kleptoparasites.Because vigilance and defensive behaviors are often costly,selection should favor hosts that adjust the amount of investmentneeded to minimize losses to kleptoparasitism. However, examplesof such facultative responses are rare. Here, we investigatethe response of cooperatively breeding pied babblers (Turdoidesbicolor) to the drongo (Dicrurus adsimilis), an avian kleptoparasitethat regularly follows pied babbler groups, often giving alarmcalls to alert the group to predators but also occasionallygiving false alarm calls in order to steal food items. We showthat pied babbler response to drongos varies markedly accordingto babbler group size. In small groups, where there are fewindividuals available to act as sentinels, babblers sentinelless when a drongo is present and respond strongly to drongoalarm calls. However, in large groups, where there are manyindividuals available to participate in predator vigilance,babblers sentinel more often when a drongo is present, rarelyrespond to drongo alarm calls, and aggressively displace drongos,with a consequent decline in the number of successful kleptoparasitismevents. This behavior represent a facultative response to akleptoparasite according to the costs versus benefits of toleratingtheir presence.     

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts

Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l⁻¹, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload-sucrose reactions. These products were purified and structurally characterized by MS-ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α-(1→1), α-(1→3) and α-(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l⁻¹, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l⁻¹ of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.     

Glucose-6-phosphate dehydrogenase deficiency in northern Mexico and description of a novel mutation

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme pathology in humans; it is X-linked inherited and causes neonatal hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the northern region of Mexico, which is important because of the genetic heterogeneity described in Mexican population. Therefore, samples from the northern Mexico were biochemically screened for G6PD-deficiency, and PCR-RFLPs, and DNA sequencing used to identify mutations in positive samples. The frequency of G6PD deficiency in the population was 0.95% (n = 1993); the mutations in 86% of these samples were G6PD A^?202A/376G , G6PD A^?376G/968C and G6PD Santamaria^376G/542T . Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country (P = 0.48336), and the unique exception with high frequency of G6PD deficiency does not involve a coastal population (Chihuahua: 2.4%). Analysis of eight polymorphic sites showed only 10 haplotypes. In one individual we identified a new G6PD mutation named Mexico DF^193A>G (rs199474830), which probably results in a damaging functional effect, according to PolyPhen analysis. Proteomic impact of the mutation is also described.     

Yeast ribosomal stalk heterogeneity in vivo shown by two-photon FCS and molecular brightness analysis

The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1α, P1β, P2α, and P2β. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1α, P1β, P2α, and P2β. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.     

Internal translation initiation on the foot-and-mouth disease virus IRES is affected by ribosomal stalk conformation

A human cell line, in which expression of the ribosomal stalk proteins P1 and P2 has been suppressed by RNAi technology, has been used to test how the loss of these proteins affects IRES-dependent translation. Foot-and-mouth disease virus (FMDV) IRES-dependent translation from a bicistronic construct is about three fold higher in the P1/P2-depleted cells than in control cells in the presence of Lb protease. By contrast, no effect on Hepatitis C virus (HCV) IRES translation was observed. These results emphasize the functional heterogeneity of the IRES and they highlight a functional connection between the ribosomal stalk and picornavirus IRES-dependent translation.