The importance of intrinsic disorder for protein phosphorylation

Reversible protein phosphorylation provides a major regulatory mechanism in eukaryotic cells. Due to the high variability of amino acid residues flanking a relatively limited number of experimentally identified phosphorylation sites, reliable prediction of such sites still remains an important issue. Here we report the development of a new web-based tool for the prediction of protein phosphorylation sites, DISPHOS (DISorder-enhanced PHOSphorylation predictor, http://www.ist.temple. edu/DISPHOS). We observed that amino acid compositions, sequence complexity, hydrophobicity, charge and other sequence attributes of regions adjacent to phosphorylation sites are very similar to those of intrinsically disordered protein regions. Thus, DISPHOS uses position-specific amino acid frequencies and disorder information to improve the discrimination between phosphorylation and non-phosphorylation sites. Based on the estimates of phosphorylation rates in various protein categories, the outputs of DISPHOS are adjusted in order to reduce the total number of misclassified residues. When tested on an equal number of phosphorylated and non-phosphorylated residues, the accuracy of DISPHOS reaches 76% for serine, 81% for threonine and 83% for tyrosine. The significant enrichment in disorder-promoting residues surrounding phosphorylation sites together with the results obtained by applying DISPHOS to various protein functional classes and proteomes, provide strong support for the hypothesis that protein phosphorylation predominantly occurs within intrinsically disordered protein regions.     

CK2 regulates in vitro the activity of the yeast cyclin-dependent kinase inhibitor Sic1

We have previously demonstrated that the cyclin-dependent kinase inhibitor (Cki) Sic1 of Saccharomyces cerevisiae is phosphorylated in vitro by the CK2 kinase on Ser(201) residue. Moreover, we have collected evidence showing that Sic1 is functionally and structurally related to mammalian Cki p27(Kip1) and binds to the mammalian Cdk2/cyclin A complex with a similar mode of inhibition. In this paper, we use SPR analysis to investigate the binding of Sic1 to the catatytic and regulatory subunits of CK2. Evidence is presented showing that phosphorylation of Sic1 at the CK2 consensus site QES(201)EDEED increases the binding of a Sic1-derived peptide to the Cdk2/cyclin A complex, a functional homologue of the yeast Cdk1/Clb5,6. Moreover, Sic1 fully phosphorylated in vitro on Ser(201) by CK2 is shown to be a stronger inhibitor of the Cdk/cyclin complexes than the unphosphorylated protein. Taken together, these data disclose the possibility that CK2 plays a role in the regulation of Sic1 activity.     

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.     

DisProt: a database of protein disorder

The Database of Protein Disorder (DisProt) is a curated database that provides structure and function information about proteins that lack a fixed three-dimensional (3D) structure under putatively native conditions, either in their entirety or in part. Starting from the central premise that intrinsic disorder is an important structural class of protein and in order to meet the increasing interest thereof, DisProt is aimed at becoming a central repository of disorder-related information. For each disordered protein, the database includes the name of the protein, various aliases, accession codes, amino acid sequence, location of the disordered region(s), and methods used for structural (disorder) characterization. If applicable, most entries also list the biological function(s) of each disordered region, how each region of disorder is used for function, as well as provide links to PubMed abstracts and major protein databases. AVAILABILITY: www.disprot.org     

Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.     

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.     

A moonlighting metabolic protein influences repair at DNA double-stranded breaks

Catalytically active proteins with divergent dual functions are often described as ‘moonlighting’. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair.