29 research outputs found
Kinetic mechanism of human dUTPase, an essential nucleotide pyrophosphatase enzyme
Human dUTPase is essential in controlling relative cellular levels of dTTP/ dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize gamma-(32) P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway
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
Antimycobacterial activity of peptide conjugate of pyridopyrimidine derivative against Mycobacterium tuberculosis in a series of in vitro and in vivo models
New pyridopyrimidine derivatives were defined using a novel HTS in silico docking method
(FRIGATE). The target protein was a dUTPase enzyme (EC 3.6.1.23; Rv2697) which plays a key
role in nucleotide biosynthesis of Mycobacterium tuberculosis (Mtb). Top hit molecules were
assayed in vitro for their antimycobacterial effect on Mtb H37Rv culture. In order to enhance the
cellular uptake rate, the TB820 compound was conjugated to a peptid-based carrier and a
nanoparticle type delivery system (polylactide-co-glycolide, PLGA) was applied. The conjugate had
relevant in vitro antitubercular activity with low in vitro and in vivo toxicity. In a Mtb H37Rv
infected guinea pig model the in vivo efficacy of orally administrated PLGA encapsulated
compound was proved: animals maintained a constant weight gain and no external clinical signs of
tuberculosis were observed. All tissue homogenates from lung, liver and kidney were found
negative for Mtb, and diagnostic autopsy showed that no significant malformations on the tissues
occurred
Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis
New semisynthetic vinca alkaloids: chemical, biochemical and cellular studies
A new semisynthetic anti-tumour bis-indol compound, KAR-2 [3′-(β-chloroethyl)-2′,4′-dioxo-3,5′-spiro-oxazolidino-4-deacetoxy-vinblastine] with lower toxicity than vinca alkaloids used in chemotherapy binds to calmodulin but, in contrast to vinblastine, does not exhibit anti-calmodulin activity. To investigate whether the modest chemical modification of bis-indol structure is responsible for the lack of anti-calmodulin potency and for the different pharmacological effects, new derivatives have been synthesized for comparative studies. The synthesis of the KAR derivatives are presented. The comparative studies showed that the spiro-oxazolidino ring and the substitution of a formyl group to a methyl one were responsible for the lack of anti-calmodulin activities. The new derivatives, similar to the mother compounds, inhibited the tubulin assembly in polymerization tests in vitro, however their inhibitory effect was highly dependent on the organization state of microtubules; bundled microtubules appeared to be resistant against the drugs. The maximal cytotoxic activities of KAR derivatives in in vivo mice hosting leukaemia P388 or Ehrlich ascites tumour cells appeared similar to that of vinblastine or vincristine, however significant prolongation of life span could be reached with KAR derivatives only after the administration of a single dose. These studies plus data obtained using a cultured human neuroblastoma cell line showed that KAR compounds displayed their cytotoxic activities at significantly higher concentrations than the mother compounds, although their antimicrotubular activities were similar in vitro. These data suggest that vinblastine/vincristine damage additional crucial cell functions, one of which could be related to calmodulin-mediated processes. © 1999 Cancer Research Campaig
Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide
Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+)-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.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 Ca(2+)-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+)-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 (4)SE(5); (7)TP(8); and (31)LS(32)). The cleavage between the (31)LS(32) peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase
Proteins with Complex Architecture as Potential Targets for Drug Design: A Case Study of Mycobacterium tuberculosis
Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel drug targets and drugs with new mechanisms of action. However, further insights are needed to establish automated protocols for target selection based on the available complete genome sequences. In the present study, we perform complete proteome level comparisons between M. tuberculosis, mycobacteria, other prokaryotes and available eukaryotes based on protein domains, local sequence similarities and protein disorder. We show that the enrichment of certain domains in the genome can indicate an important function specific to M. tuberculosis. We identified two families, termed pkn and PE/PPE that stand out in this respect. The common property of these two protein families is a complex domain organization that combines species-specific regions, commonly occurring domains and disordered segments. Besides highlighting promising novel drug target candidates in M. tuberculosis, the presented analysis can also be viewed as a general protocol to identify proteins involved in species-specific functions in a given organism. We conclude that target selection protocols should be extended to include proteins with complex domain architectures instead of focusing on sequentially unique and essential proteins only