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941.
Kamal Akhtar Vibhor Gupta Anita Koul Neelima Alam Rajiv Bhat Rameshwar N. K. Bamezai 《The Journal of biological chemistry》2009,284(18):11971-11981
In this study, we attempted to understand the mechanism of regulation of
the activity and allosteric behavior of the pyruvate kinase M2
enzyme and two of its missense mutations, H391Y and K422R, found in cells from
Bloom syndrome patients, prone to develop cancer. Results show that despite
the presence of mutations in the intersubunit contact domain, the K422R and
H391Y mutant proteins maintained their homotetrameric structure, similar to
the wild-type protein, but showed a loss of activity of 75 and 20%,
respectively. Interestingly, H391Y showed a 6-fold increase in affinity for
its substrate phosphoenolpyruvate and behaved like a non-allosteric protein
with compromised cooperative binding. However, the affinity for
phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y
showed enhanced thermal stability, stability over a range of pH values, a
lesser effect of the allosteric inhibitor Phe, and resistance toward
structural alteration upon binding of the activator (fructose
1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in
the pH optimum from 7.4 to 7.0. Although this study signifies the importance
of conserved amino acid residues in long-range communications between the
subunits of multimeric proteins, the altered behavior of mutants is suggestive
of their probable role in tumor-promoting growth and metabolism in Bloom
syndrome patients with defective pyruvate kinase M2.Pyruvate kinase
(PK3; EC 2.7.1.40), a
pacemaker of the glycolytic pathway, catalyzes irreversibly the
transphosphorylation from P-enolpyruvate to ADP, generating pyruvate and ATP
(1,
2). There are four different
isozymes (L, R, M1, and M2) in mammalian tissues, which
differ in their regulatory properties. These isozymes are allosteric in nature
with the exception of the M1 form, present in skeletal muscle and
brain
(3–7).
PKM2 is a ubiquitous prototype enzyme present in all tissues during
the embryonic stage and is gradually replaced by other isozymic forms in
specific tissues during development. The M2, L, and R isozymes show
homotropic cooperative activation with P-enolpyruvate and heterotropic
cooperative activation with Fru-1,6-P2
(8–10).
The M1 isozyme is regulated by neither P-enolpyruvate nor
Fru-1,6-P2 because of its intrinsic active conformation in the
R-state (5,
6). Under unfavorable
conditions such as hypoxia and lack of glucose supply, the anaerobic tissues
and tumor cells rely heavily on PKM2 for ATP production
(7). Therefore, stringent
control of PK activity is of great importance not only for cell metabolism but
also for tumorigenic proliferation.The M1 and M2 isozymes are produced from a single
gene locus by mutually exclusive alternative splicing
(11–14).
In the human M1 and M2 isozymes, the exon that is
exchanged because of alternative splicing encodes 56 amino acids, in which a
total of 22 amino acids differ within a length of 45 residues. The residues
located in this region form the major intersubunit contact domain
(8). The distinguishable
kinetic properties of the M1 and M2 isozymes are
attributed to these amino acid substitutions. It has been shown by x-ray
crystallographic analyses and computer modeling that the corresponding regions
of their polypeptides participate directly in the intersubunit contact, which
is responsible for the intersubunit communication required for allosteric
cooperativity (8,
15).PK has been largely conserved throughout evolution. The enzyme is usually a
homotetramer composed of four identical subunits, and each subunit consists of
four domains: the A-, B-, and C-domains and the N-terminal domain. The
structure of human PKM2 was recently determined in complex with
inhibitors (16). In mammalian
cells, PK activity is regulated by two different mechanisms: one at the level
of expression and the other through allosteric regulation. The catalytic site
usually composes a small part of the enzyme, but allosteric control is
transmitted over a long range, thus increasing the number of possible residues
involved in regulation. The allosteric transition in PK involves mutual
rotations of the A- and C-domains within each subunit and the subunit within
the tetramer (14). The
residues at the subunit interfaces have the critical function of relaying the
allosteric signal from and to the catalytic and regulatory sites. This region
also transmits the allosteric signal between P-enolpyruvate- and
Fru-1,6-P2-binding sites. Despite the availability of structural
details of several PK isozymes, it is difficult to identify the structural
elements that play an important role in PK regulation and propagation of the
allosteric signals. Although the role of some of the PK residues (positions
340, 389, 398, 401, 402, 408, 423, and 427) has been studied in allosteric
regulation (10,
17–19)
by in vitro site-directed mutagenesis, the absence of these mutations
in any naturally occurring condition presents limitations in attributing a
biological role to the introduced changes.The natural mutations H391Y and K422R (reported previously as K421R) were
reported by us for the first time in the PKM2 gene in a Bloom
syndrome cell line and in the lymphocytes of an Indian Bloom syndrome patient,
respectively (20). The two
missense mutations, located in the region of the intersubunit contact domain
(Fig. 1, A and
B), presented with the biochemical phenotype of
down-regulated enzyme activity to different extents
(20) and were expected to
influence the allosteric nature of the enzyme. The regulatory behavior of
allosteric PK has been described by a two-state model that proposes an active
(R) and an inactive (T) form of the macromolecule with differential affinity
for ligands (15). Upon binding
of the substrate or its analogs, the enzyme undergoes a transition from a low
activity/low affinity conformation (T state) to a high activity/high affinity
conformation (R state). The binding of phenylalanine produces a global
structural change and exhibits reduced affinity for substrate P-enolpyruvate
in the T state
(21–23).
Previous studies have demonstrated that each individual domain acts as a rigid
body and that, upon transition from the T to the R state, the domain of the
functional tetramer modifies its relative orientation by 29°. These
movements bring conformational change to the active site, which, upon
transition to the T state, undergoes a distortion of the
P-enolpyruvate-binding site
(24).Open in a separate windowFIGURE 1.A, ribbon diagram of the overall structure of PK showing the
positions of the two mutations, H391Y and K422R, along with the active site
and Fru-1,6-P2-binding site. B, intersubunit contact
domain of PK. The major amino acid residues and side chains at the tetramer
interface region are shown.Because the mutations observed by us previously
(20) are located at highly
conserved positions not only in different isozymic forms but also across the
species (supplemental Fig. S1) and are observed in the genetic background of a
syndrome prone to cancer in early age, a study related to the
structure-function correlations of these mutations is likely to provide
insight into their possible biological importance, especially in the context
of recent research highlighting the importance of PKM2 in tumor
promotion and growth. In this study, we investigated the role of the two
natural missense mutations, after site-directed mutagenesis in the
PKM2 gene, in the regulation of allosteric properties as well as
their effects on the secondary and tertiary structures in comparison with
wild-type PKM2 (PK-WT). An attempt has also been made to understand
the effects of these mutations at the interface of the subunits on the signal
transmission pathway within the protein. 相似文献
942.
Background HLA-DQ alleles are involved in the pathogenesis of hypersensitivity reactions, with HLA-DQ8 associated with several human autoimmune disorders. Limited success has been achieved using sequence-based computational techniques for predicting HLA-DQ8-restricted T cell epitopes while accuracy and efficiency of recently developed structure-based models need to be improved. Results We describe a combined structure-based prediction approach for DQ8-restricted T cell epitope prediction using a recently developed fast and accurate docking protocol, pDOCK, and molecular surface electrostatic potential (MSEP)-based clustering of pMHC binding interfaces. The prediction model was rigorously trained, tested and validated using experimentally verified DQ8 binding and non-binding peptides. High prediction accuracy (average area under the ROC curve, average AROC>0.94) is validated against experimental data. Our model also predicts all binding registers correctly and known T cell activators with 77% accuracy. We also studied the patterns of DQ8-binding peptides and reassure the existence of epitopes not conforming to binding motifs. Conclusions We have developed a model that can be successfully applied as a generic protocol for easy in silico identification of potential immunogenic T cell epitopes. The current model is therefore applicable for screening vaccine candidates irrespective of sequence motifs. We have also illustrated efficient discrimination of different categories of binders from non-binders as well as different categories of pMHC agonists from non-agonists, while accurately predicting the binding registers of DQ8-restricted peptides. This combined approach provides a set of sensitive and specific computational tools to facilitate high-throughput screening of peptides for immunotherapeutic applications such as controlling allergic and autoimmune responses. 相似文献
943.
To clarify the relationship between cultivar difference in the sensitivity of net photosynthesis to ozone (O(3) ) and the reactive oxygen species (ROS) scavenging system in wheat (Triticum aestivum), we investigated the effects of chronic exposure to ambient levels of O(3) on gas exchange rates, activity and concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), activity of ROS scavenging enzymes and concentration of antioxidants of the flag leaf in two Japanese winter wheat cultivars (Norin 61 and Shirogane-komugi). Although the net photosynthetic rate of the flag leaf in Norin 61 was not significantly reduced by exposure to O(3) , that in Shirogane-komugi was significantly reduced by the exposure to O(3) during the anthesis and early grain-filling stages. In the two cultivars, stomatal diffusive conductance to H(2) O of the flag leaf was not significantly affected by the exposure to O(3) . The exposure to O(3) induced significant reductions in the activity and concentration of Rubisco, activities of catalase (CAT) and monodehydroascorbate reductase (MDAR) and concentrations of reduced form of ascorbate and total glutathione of the flag leaf in Shirogane-komugi. It was concluded that the sensitivity of net photosynthesis of flag leaf to O(3) is higher in Shirogane-komugi than in Norin 61, and the difference in the sensitivity to O(3) between the two cultivars is mainly due to that in the effects of O(3) on the detoxification ability of ROS, mainly determined by the activity of ROS scavenging enzymes, such as CAT and MDAR. 相似文献
944.
Khan A Vaibhav K Javed H Moshahid Khan M Tabassum R Ahmed ME Srivastava P Khuwaja G Islam F Saeed Siddiqui M Shafi MM Islam F 《Molecular and cellular biochemistry》2012,369(1-2):55-65
Beta-amyloid (Aβ) peptides are considered to play a major role in the pathogenesis of Alzheimer's disease (AD) and compounds that can prevent pathways of Aβ-induced neurotoxicity may be potential therapeutic agents for treatment of AD. This study examined the hypothesis that thymoquinone (TQ) would reduce oxidative stress and mitochondrial dysfunction in differentiated pheochromocytoma (PC 12) cells exposed to Aβ fragment 25-35 (Aβ(25-35)). To test this hypothesis, Aβ was used to induce an in vitro model of AD in differentiated PC 12 cell line of rat. After 24?h of exposure with Aβ(25-35), a significant reduction in cell viability and mitochondrial membrane potential (MMP) was observed. In addition, a significant elevation in the TBARS content and nitric oxide (NO) and activity of acetylcholine esterase (AChE) was observed which was restored significantly by TQ pretreatment. Furthermore, TQ also ameliorated glutathione and its dependent enzymes (glutathione peroxidase, glutathione reductase) which were depleted by Aβ(25-35) in PC 12 cells. These results were supported by the immunocytochemical finding that has shown protection of cells by TQ from noxious effects of Aβ(25-35). These results indicate that TQ holds potential for neuroprotection and may be a promising approach for the treatment of neurodegenerative disorders including AD. 相似文献
945.
946.
Vijay K. Tiwari Oscar Riera-Lizarazu Hilary L. Gunn KaSandra Lopez M. Javed Iqbal Shahryar F. Kianian Jeffrey M. Leonard 《PloS one》2012,7(11)
Physical mapping and genome sequencing are underway for the ≈17 Gb wheat genome. Physical mapping methods independent of meiotic recombination, such as radiation hybrid (RH) mapping, will aid precise anchoring of BAC contigs in the large regions of suppressed recombination in Triticeae genomes. Reports of endosperm development following pollination with irradiated pollen at dosages that cause embryo abortion prompted us to investigate endosperm as a potential source of RH mapping germplasm. Here, we report a novel approach to construct RH based physical maps of all seven D-genome chromosomes of the hexaploid wheat ‘Chinese Spring’, simultaneously. An 81-member subset of endosperm samples derived from 20-Gy irradiated pollen was genotyped for deletions, and 737 markers were mapped on seven D-genome chromosomes. Analysis of well-defined regions of six chromosomes suggested a map resolution of ∼830 kb could be achieved; this estimate was validated with assays of markers from a sequenced contig. We estimate that the panel contains ∼6,000 deletion bins for D-genome chromosomes and will require ∼18,000 markers for high resolution mapping. Map-based deletion estimates revealed a majority of 1–20 Mb interstitial deletions suggesting mutagenic repair of double-strand breaks in pollen provides a useful resource for RH mapping and map based cloning studies. 相似文献
947.
A synthetic pyruvate:H2 pathway was constructed in Escherichia coli BL21(DE3) by co-expression of six proteins: E. coli YdbK, Clostridium pasteurianum [4Fe–4S]-ferredoxin, and Clostridium acetobutylicum HydF, HydE, HydG, and HydA. The effect of cofactor addition and host strain on H2 yield and fermentation product accumulation was studied, together with in vitro reconstitution of the entire pathway. The deletion of iscR and/or the addition of thiamine pyrophosphate to the medium enhanced the total and specific activity of recombinant YdbK and increased the yield of H2 per glucose. It was concluded that the introduced pathway outcompeted other pyruvate-consuming reactions, and that the ability to compete for pyruvate at least in part was determined by total YdbK activity. The results demonstrate the successful construction of a high-yielding H2 pathway in a microorganism that effectively does not synthesize any H2. The additional co-expression of Bacillus subtilis AmyE enabled starch-dependent H2 synthesis in minimal media. 相似文献
948.
949.
Though many microorganisms that are capable of using phenol as sole sourceof carbon have been isolated and characterized, only a few organisms degradingsubstituted phenols have been described to date. In this study, one strain ofmicroorganism that is capable of using phenol (3000 ppm), 4-aminophenol(4000 ppm) and 4-acetamidophenol (4000 ppm) as sole source of carbon andenergy was isolated and characterized. This strain was obtained by enrichmentculture from a site contaminated with compounds like 4-acetamidophenol,4-aminophenol and phenol in Pakistan at Bhai Pheru. The contaminated siteis able to support large bacterial community as indicated by the viable cellcounts (2 × 104–5 × 108) per gram of soil. Detailed taxonomic studies identified the organisms as Pseudomonas species designated as strain STI. The isolate also showed growth on other organic compounds like aniline, benzene, benzyl alcohol, benzyl bromide, toluene, -cresol, trichloroethylene and o-xylene. Optimum growth temperature and pH were found to be 30 °C and 7, respectively, while growth at 4, 25 and 35 °C and at pH 8 and 9 was also observed. Non growing suspended cells of strain ST1 degraded 68, 96 and 76.8% of 4-aminophenol (1000 ppm), phenol (500 ppm) and 4-acetamidophenol (1000 ppm), respectively, in 72 hrs. The isolation and characterization of Pseudomonas speciesstrain ST1, may contribute to efforts on phenolic bioremediation, particularly in anenvironment with very high levels of 4-acetamidophenol and 4-aminophenol. 相似文献
950.