66 research outputs found
Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control
Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium
Proteins responsible for the integrity of the genome are often used targets in drug therapies against various diseases. The inhibitors of these proteins are also important to study the pathways in genome integrity maintenance. A prominent example is Ugi, a well known cross-species inhibitor protein of the enzyme uracil-DNA glycosylase, responsible for uracil excision from DNA. Here, we report that a Staphylococcus pathogenicity island repressor protein called StlSaPIbov1 (Stl) exhibits potent dUTPase inhibition in Mycobacteria. To our knowledge, this is the first indication of a cross-species inhibitor protein for any dUTPase. We demonstrate that the Staphylococcus aureus Stl and the Mycobacterium tuberculosis dUTPase form a stable complex and that in this complex, the enzymatic activity of dUTPase is strongly inhibited. We also found that the expression of the Stl protein in Mycobacterium smegmatis led to highly increased cellular dUTP levels in the mycobacterial cell, this effect being in agreement with its dUTPase inhibitory role. In addition, Stl expression in M. smegmatis drastically decreased colony forming ability, as well, indicating significant perturbation of the phenotype. Therefore, we propose that Stl can be considered to be a cross-species dUTPase inhibitor and may be used as an important reagent in dUTPase inhibition experiments either in vitro/in situ or in vivo
dUTPase based switch controls transfer of virulence genes in order to preserve integrity of the transferred mobile genetic elements
dUTPases ubiquitously regulate cellular dUTP levels to preserve
genome integrity. Recently, several other cellular processes were
reported to be controlled by dUTPases including the horizontal
transfer of Staphylococcus aureus pathogenicity islands (SaPI).
SaPIs are mobil genetic elements that encode virulence enhancing
factors e.g. toxins. Here, phage dUTPases were proposed to
counteract the repressor protein (Stl) and promote SaPI excision
and transfer. A G protein-like mechanism was proposed which is
unexpected in light of the kinetic mechanism of dUTPase.
Here we investigate the molecular mechanism of SaPI transfer
regulation, using numerous dUTPase variants and a wide range
of in vitro methods (steady-state and transient kinetics, VIS and
fluorescence spectroscopy, EMSA, quartz crystal microbalance,
X-ray crystallography).
Our results unambiguously show that Stl inhibits the enzymatic
activity of dUTPase in the nM concentration range and
dUTP strongly inhibits the dUTPase: Stl complexation. These
results identify Stl as a highly potent dUTPase inhibitor protein
and disprove the G protein-like mechanism. Importantly, our
results clearly show that the dUTPase:dUTP complex is inaccessible
to the Stl repressor. Unlike in small GTPases, hydrolysis of
the substrate nucleoside triphosphate (dUTP in this case) is
required prior to the interaction with the partner (Stl repressor in
this case). We propose that dUTPase can efficiently interact with
Stl and induce SaPI excision only if the cellular dUTP level is low (i.e. when dUTPase resides mainly in the apo enzyme form)
while high dUTP levels would inhibit SaPI transfer. This mechanism
may serve the preservation of the integrity of the transferred
SaPI genes and links the well-known metabolic role of
dUTPases to their newly revealed regulatory function in spread
of virulence factors
Covariant description of kinetic freeze out through a finite time-like layer
The Freeze Out (FO) problem is addressed for a covariant FO probability and a
finite FO layer with a time-like normal vector continuing the line of studies
introduced in Ref. [1]. The resulting post FO momentum distribution functions
are presented and discussed. We show that in general the post FO distributions
are non-thermal and asymmetric distributions even for time-like FO situations.Comment: 10 pages, 12 figures, major rewrite with changed content, corrected
typos and new references adde
Covariant description of kinetic freeze out through a finite space-like layer
The problem of Freeze Out (FO) in relativistic heavy ion reactions is
addressed. We develop and analyze an idealized one-dimensional model of FO in a
finite layer, based on the covariant FO probability. The resulting post FO
phase-space distributions are discussed for different FO probabilities and
layer thicknesses.Comment: 16 pages, 19 figures, changed content, references adde
Evolutionary and socio-cultural influences on feelings and attitudes towards nature: a cross-cultural study
Mounting environmental issues have prompted reconsideration of the human–nature relationship. Accordingly, attitudes to nature, as an important dimension of human–nature interactions, have become a research focus. How feelings and attitudes towards nature are influenced by evolutionary and social-cultural constructions, and whether there is variation between different cultural groups, demands more attention. Using a survey of visitors to two very different National Parks, the New Forest National Park, England and Jiuzhaigou Scenic Area, China, this paper shows that of nationality and living environment, differences between the two nationalities were significant in respect of both attitudes and feelings. Specifically, it demonstrates that the biophilia thesis, which purports that people have an innate and a genetically inherited need for affiliation with nature, is influenced by their socio-cultural environment, in particular their national culture, but also by their current living place. The study contributes to our understanding of sustainable tourism in natural areas
Structural model of human dUTPase in complex with a novel proteinaceous inhibitor
Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein StlSaPIBov1 (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition. Small-angle X-ray scattering (SAXS) complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) data allowed us to obtain 3D structural models comprising a trimeric dUTPase complexed with separate Stl monomers. These models thus reveal that upon dUTPase-Stl complex formation the functional homodimer of Stl repressor dissociates, which abolishes the DNA binding ability of the protein. Active site forming dUTPase segments were directly identified to be involved in the dUTPase-Stl interaction by HDX-MS, explaining the loss of dUTPase activity upon complexation. Our results provide key novel structural insights that pave the way for further applications of the first potent proteinaceous inhibitor of human dUTPase
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