Vliv fosforylace na 3D konformaci proteinů

Fosforylace je jedna z nejrozšířenějších proteinových posttranslačních modifikací. Pochopení funkce a regulace fosforylace je významné pro diagnózu a léčbu mnoha onemocnění. Tato práce shrnuje studie analyzující fosforylace pomocí bioinformatických nástrojů na velkých datových souborech. V této práci jsou popsány obecné principy, jak fosforylace ovlivňuje fyzikálně-chemické vlastnosti proteinů. V prvních dvou kapitolách jsou nastíněny evoluční zákonitosti, funkce a regulace proteinových kináz a fosfatáz. Ve třetí kapitole se práce zaměřuje na rozložení fosforylačních míst proteinů napříč různými organizmy (rostlinnými a živočišnými). Poslední kapitola se věnuje shrnutí dnešních poznatků o alosterickém a ortosterickém efektu fosforylace a vlivu fosforylace na 2D a 3D strukturu proteinů.Phosphorylation is one of the most ubiquitous posttranslational modification types. Understanding of itsfunction and regulation has significant impact on diagnosis and treatment of many diseases. This thesis presents and summarizes the results of several publications that analyze phosphorylation on large datasets using bioinformatics tools. In this thesis the general principles how the phosphorylation influences physico-chemical properties of proteins are described. In the first and the second chapter the evolution principles, function and regulation of protein kinases and phosphatases are provided. In the third chapter thesis concentrates on the distributions of phosphorylated sites among organisms (plants and animals). In the last chapter current knowledge of orthosteric and allosteric effects of phosphorylation as well as its effects on 2D and 3D structure of phosphorylated proteins is summarized.Department of Cell BiologyKatedra buněčné biologiePřírodovědecká fakultaFaculty of Scienc

The Perseus computational platform for comprehensive analysis of (prote)omics data

A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass spectrometry-based analysis. We developed the Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical toots for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. ALL activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets

Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human a-actinin-4 (ACTN4), Tyr[superscript 4] and Tyr[superscript 31] are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr[superscript 4] site. We found that introducing a phosphomimetic mutation at only Tyr[superscript 31] was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr[superscript 31] is mostly buried and that phosphorylation of Tyr[superscript 4] would increase the solvent exposure and thus kinase accessibility of Tyr[superscript 31]. In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr[superscript 4], whereas a truncated mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at a background amount similar to that observed for a double phosphomimetic mutant of Tyr[superscript 4] and Tyr[superscript 31]. We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr[superscript 31] can be dictated by the kinase affinity for Tyr[superscript 4]. This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site.National Institutes of Health (U.S.) (Grant R01 GM69668

The role of structural disorder in cell cycle regulation, related clinical proteomics, disease development and drug targeting

Understanding the molecular mechanisms of the regulation of cell cycle is a central issue in molecular cell biology, due to its fundamental role in the existence of cells. The regulatory circuits that make decisions on when a cell should divide are very complex and particularly subtly balanced in eukaryotes, in which the harmony of many different cells in an organism is essential for life. Several hundred proteins are involved in these processes, and a great deal of studies attests that most of them have functionally relevant intrinsic structural disorder. Structural disorder imparts many functional advantages on these proteins, and we discuss it in detail that it is involved in all key steps from signaling through the cell membrane to regulating transcription of proteins that execute timely responses to an ever-changing environment. © Informa Uk, Ltd

Role post-translačních modifikací, O-GlcNAcylace a fosforylace, v neurodegenerativnách onemocněních a hypometabolismu CNS

Post-translační modifikace jsou jedny z hlavních mechanismů, které významně zvyšují variabilitu funkce proteinů. O-GlcNAcylace a fosforylace patří mezi nejrozšířenější a nejvíce studované post-translační modifikace. Za fyziologických podmínek OGlcNAcylace působí jako metabolický senzor, který spojuje metabolismus glukózy s běžnou funkcí neuronů. Reverzibilní fosforylace je jedním z mechanismů, které mohou snížit metabolismus regulováním rychlosti toku metabolickými cestami. Poruchy regulace těchto modifikací jsou spojeny s neurodegenerativními poruchami a hypometabolismem. Práce se zaměřuje na korelaci těchto dvou modifikací, jejich vzájemný vztah a dopad na neurodegenerativní onemocnění a jiné fyziologické modely. Klíčová slova: hypometabolismus, OGlcNAcylace, fosforylace, post-translační modifikace, neurodegenrativní onemocnění, hibernace, kalorická restrikce, paměť, učeníPost-translational modifications are major mechanisms that highly increase the variability in protein function. O-GlcNAcylation and phosphorylation are among the most extensively studied post-translational modifications in research to date. In physiological conditions, O- GlcNAcylation acts as a metabolic sensor that links glucose metabolism to normal neuronal functioning. Reversible phosphorylation is one of the mechanisms that can downregulate metabolism by regulating the rates of flux through metabolic pathways. The impairments in the regulation of these modifications are linked to with neurodegenerative disorders and hypometabolism. This thesis focuses on the crosstalk and correlation between these two modifications, their reciprocal relationship and their mutual impact on their regulations in models of neurodegenerative diseases and disease non-related models. Keywords: hypometabolism, O-GlcNAcylation, phosphorylation, post- translational modifications, neurodegenerative disorders, hibernation, caloric restriction, memory, learningDepartment of PhysiologyKatedra fyziologieFaculty of SciencePřírodovědecká fakult

Development and Applications of Mass Spectrometric Methods for Phosphorylation Analysis

Protein phosphorylation modification regulates numerous cellular functions by a reversible and selective control of kinases and phosphatases. To understand the entire dynamic network of phosphorylation requires sensitive and reliable quantification of phosphorylation, measurements that can be achieved by mass spectrometry. In this research, we established efficient MALDI-mass spectrometric methods as strategies for single- or multi-site phosphorylation quantification without the use of isotopes, chromatography and calibration curves. The methods were assessed by analyzing peptide standards with different single-multiple phosphorylation sites, showing a wide dynamic range, good accuracy and reproducibility. This is the first label-free MALDI method without using a calibration methodology proposed for quantification of in vitro phosphorylation in a kinase assay. Moreover, advanced mass spectrometry empowers identification of a highly conserved Cdk2 phosphorylation site of HIV-1 reverse transcriptase (RT) at Thr 261 across thousands of HIV-1 strains. We demonstrated phosphorylation on HIV-1 RT peptides and protein in in vitro assays, and confirmed phosphorylation in vivo with antibodies and mutation studies. Blocking this phosphorylation by p21, a naturally occurring Cdk inhibitor, defines a potential Cdk2-mediated cell-intrinsic mechanism for restricting HIV-replication in a clinically significant way

Phosphorylation variation during the cell cycle scales with structural propensities of proteins.

Phosphorylation at specific residues can activate a protein, lead to its localization to particular compartments, be a trigger for protein degradation and fulfill many other biological functions. Protein phosphorylation is increasingly being studied at a large scale and in a quantitative manner that includes a temporal dimension. By contrast, structural properties of identified phosphorylation sites have so far been investigated in a static, non-quantitative way. Here we combine for the first time dynamic properties of the phosphoproteome with protein structural features. At six time points of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels, whereas regions with predominantly regular secondary structures retain more constant phosphorylation levels. The two groups show preferences for different amino acids in their kinase recognition motifs-proline and other disorder-associated residues are enriched in the former group and charged residues in the latter. Furthermore, these preferences scale with the degree of disorderedness, from regular to irregular and to disordered structures. Our results suggest that the structural organization of the region in which a phosphorylation site resides may serve as an additional control mechanism. They also imply that phosphorylation sites are associated with different time scales that serve different functional needs