Gergely Róna

Structural Biology and Regulation of Protein Import into the Nucleus

Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g., importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways.

Uracil-containing DNA in Drosophila: stability, stage-specific accumulation, and developmental involvement.

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade.

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

Calcium-induced tripartite binding of intrinsically disordered calpastatin to its cognate enzyme, calpain

MINT‐6549073: Calpain-2 catalytic subunit (uniprotkb:P04632), Calpain-2 catalytic subunit (uniprotkb:P17655) and calpastatin (uniprotkb:P20810) physically interact (MI:0218) by nuclear magnetic resonance (MI:0077)

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation.

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.

Structure and enzymatic mechanism of a moonlighting dUTPase

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. In Staphylococcus, phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Å resolution three-dimensional structure of a ϕ11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small β-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of ϕ11 phage dUTPase.

Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.

NLS copy-number variation governs efficiency of nuclear import--case study on dUTPases.

Nucleocytoplasmic trafficking of large macromolecules requires an active transport machinery. In many cases, this is initiated by binding of the nuclear localization signal (NLS) peptide of cargo proteins to importin‐α molecules. Fine orchestration of nucleocytoplasmic trafficking is of particularly high importance for proteins involved in maintenance of genome integrity, such as dUTPases, which are responsible for prevention of uracil incorporation into the genome. In most eukaryotes, dUTPases have two homotrimeric isoforms: one of these contains three NLSs and is present in the cell nucleus, while the other is located in the cytoplasm or the mitochondria. Here we focus on the unusual occurrence of a pseudo‐heterotrimeric dUTPase in Drosophila virilis that contains one NLS, and investigate its localization pattern compared to the homotrimeric dUTPase isoforms of Drosophila melanogaster. Although the interaction of individual NLSs with importin‐α has been well characterized, the question of how multiple NLSs of oligomeric cargo proteins affect their trafficking has been less frequently addressed in adequate detail. Using the D. virilis dUTPase as a fully relevant physiologically occurring model protein, we show that NLS copy number influences the efficiency of nuclear import in both insect and mammalian cell lines, as well as in D. melanogaster and D. virilis tissues. Biophysical data indicate that NLS copy number determines the stoichiometry of complexation between importin‐α and dUTPases. The main conclusion of our study is that, in D. virilis, a single dUTPase isoform efficiently reproduces the cellular dUTPase distribution pattern that requires two isoforms in D. melanogaster.

Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

Background Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca2+-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase. Methodology/Principal Findings Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca2+-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca2+-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues 4SE5; 7TP8; and 31LS32). The cleavage between the 31LS32 peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization. Conclusions/Significance Results argue for a mechanism where Ca2+-calpain may regulate nuclear availability and degradation of dUTPase.

Crystallization and preliminary crystallographic analysis of dUTPase from the φ11 helper phage of Staphylococcus aureus.

Staphylococcus aureus superantigen-carrying pathogenicity islands (SaPIs) play a determinant role in spreading virulence genes among bacterial populations that constitute a major health hazard. Repressor (Stl) proteins are responsible for the transcriptional regulation of pathogenicity island genes. Recently, a derepressing interaction between the repressor Stl SaPIbov1 and dUTPase from the φ11 helper phage has been suggested [Tormo-Más et al. (2010), Nature (London), 465, 779-782]. Towards elucidation of the molecular mechanism of this interaction, this study reports the expression, purification and X-ray analysis of φ11 dUTPase, which contains a phage-specific polypeptide segment that is not present in other dUTPases. Crystals were obtained using the hanging-drop vapour-diffusion method at room temperature. Data were collected to 2.98 Å resolution from one type of crystal. The crystal of φ11 dUTPase belonged to the cubic space group I23, with unit-cell parameters a = 98.16 Å, α = β = γ = 90.00°.