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881.
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
882.
883.
S. Farooq I. Hussain M.A. Mir M.A. Bhat S.A. Wani 《Letters in applied microbiology》2009,48(6):692-697
Aims: To study the prevalence and characterize atypical enteropathogenic Escherichia coli (EPEC) and Shiga toxin producing E. coli (STEC) in avian species in India. Methods and Results: Two hundred and twelve faecal samples collected from 62 chickens, 50 ducks and 100 pigeons were investigated for the presence of stx1, stx2, eae and ehxA virulence genes by multiplex PCR. In all, 42 E. coli isolates (25 chicken, 2 duck and 15 pigeon) possessed at least one virulence gene. Out of these, nine (4·24%) isolates were STEC and 33 (15·56%) were EPEC. All isolates from duck and chicken were EPEC while among 15 pigeon isolates nine (60%) were STEC and six (40%) were EPEC. Among the STEC isolates four each carried stx1 or stx2 and one possessed both stx1 and stx2. Subtype analysis of stx revealed the presence of stx2f in four STEC isolates. None of the STEC isolates carried stx1c, stx2c, stx2d or stx2e. Isolates carrying stx2f demonstrated vero cell toxicity. One each belonged to serogroup O17 and O78, while one was rough and the other untypeable. All EPEC isolates were atypical as they lacked bfpA. This appears to be the first report of detection of stx2f from India. Conclusions: The study established the presence of stx1 and stx2f containing E. coli in pigeons and atypical EPEC in poultry in India. Pigeons might serve as vectors for transmission of STEC to environment and humans. Significance and Impact of the Study: Taking into account the close contact between fanciers and pigeons, these findings warrant a more critical appraisal of these zoonotic pathogens in pigeons and humans. 相似文献
884.
Aurelia Sima Maxime Parisotto Sylvie Mader Pangala V. Bhat 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009,1790(12):1660-1664
Background
Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis.Methods
RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography.Results
RALDH3 oxidized all-trans retinal with high catalytic efficiency (Vmax/Km = 77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (Vmax/Km = 27.4), and oxidized 13-cis retinal with lower catalytic efficiency (Vmax/Km = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl2 activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity.Conclusions
These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.General significance
The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues. 相似文献885.
Karunakaran M Mondal M Rajarajan K Karmakar HD Bhat BP Das J Bora B Baruah KK Rajkhowa C 《Animal reproduction science》2009,111(1):112-119
Male Naga pig of India, a miniature breed is known for its meat quality and early puberty. No scientific efforts were made to verify the farmers' view that this breed reaches puberty at around 2 months of age. A preliminary study was, therefore, conducted with the objectives: (a) to find out the age at puberty based on mature spermiogram and in vivo pregnancy and (b) to record the sperm morphology in different parts of the epididymis. Animals were selected from two different age groups: group I aged 53 days and 2.4 kg and group II of 85 days and 3.0 kg. Semen samples collected from different sections of epididymis were analyzed for sperm motility, live spermatozoa, and morphological abnormalities. Motility increased (P<0.01) and live spermatozoa and total morphological abnormalities decreased (P<0.001) from caput through cauda epididymis in both the groups. Sperm motility, live spermatozoa and morphologically normal spermatozoa in each section of the epididymis were higher (P<0.01) in group II than I. Boars with >60% progressive motility, >70% live spermatozoa, <15% total morphological abnormalities and <10% abnormal acrosomes in cauda epididymal spermatozoa were considered mature spermiogram. As per this definition, pigs of group II had only mature spermiogram. In vivo pregnancy confirmation indicated that Naga boar could impregnate female as early as 90 days of age. In conclusion, Naga boar attained puberty by not later than 3 months with 3.0 kg, which is the lowest body weight at puberty in this species reported so far, as reflected by mature epididymal spermiogram and in vivo pregnancy confirmation. 相似文献
886.
Van Buskirk GA Arsulowicz D Basu P Block L Cai B Cleary GW Ghosh T González MA Kanios D Marques M Noonan PK Ocheltree T Schwarz P Shah V Spencer TS Tavares L Ulman K Uppoor R Yeoh T 《AAPS PharmSciTech》2012,13(1):218-230
In this whitepaper, the Manufacturing Technical Committee (MTC) of the Product Quality Research Institute has updated the 1997 Transdermal Drug Delivery Systems Scale-Up and Post Approval Change workshop report findings to add important new product development and control principles. Important topics reviewed include ICH harmonization, quality by design, process analytical technologies, product and process validation, improvements to control of critical excipients, and discussion of Food and Drug Administration's Guidance on Residual Drug in Transdermal and Related Drug Delivery Systems as well as current thinking and trends on in vitro-in vivo correlation considerations for transdermal systems. 相似文献
887.
Venkatraman J Bhat J Solapure SM Sandesh J Sarkar D Aishwarya S Mukherjee K Datta S Malolanarasimhan K Bandodkar B Das KS 《Journal of biomolecular screening》2012,17(3):293-302
The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays. 相似文献
888.
Sujay V Gowda MV Pandey MK Bhat RS Khedikar YP Nadaf HL Gautami B Sarvamangala C Lingaraju S Radhakrishan T Knapp SJ Varshney RK 《Molecular breeding : new strategies in plant improvement》2012,30(2):773-788
Late leaf spot (LLS) and rust have the greatest impact on yield losses worldwide in groundnut (Arachis hypogaea L.). With the objective of identifying tightly linked markers to these diseases, a total of 3,097 simple sequence repeats (SSRs) were screened on the parents of two recombinant inbred line (RIL) populations, namely TAG 24?×?GPBD 4 (RIL-4) and TG 26?×?GPBD 4 (RIL-5), and segregation data were obtained for 209 marker loci for each of the mapping populations. Linkage map analysis of the 209 loci resulted in the mapping of 188 and 181 loci in RIL-4 and RIL-5 respectively. Using 143 markers common to the two maps, a consensus map with 225 SSR loci and total map distance of 1,152.9?cM was developed. Comprehensive quantitative trait locus (QTL) analysis detected a total of 28 QTL for LLS and 15 QTL for rust. A major QTL for LLS, namely QTL(LLS)01 (GM1573/GM1009-pPGPseq8D09), with 10.27-62.34% phenotypic variance explained (PVE) was detected in all the six environments in the RIL-4 population. In the case of rust resistance, in addition to marker IPAHM103 identified earlier, four new markers (GM2009, GM1536, GM2301 and GM2079) showed significant association with the major QTL (82.96% PVE). Localization of 42 QTL for LLS and rust on the consensus map identified two candidate genomic regions conferring resistance to LLS and rust. One region present on linkage group AhXV contained three QTL each for LLS (up to 67.98% PVE) and rust (up to 82.96% PVE). The second candidate genomic region contained the major QTL with up to 62.34% PVE for LLS. Molecular markers associated with the major QTL for resistance to LLS and rust can be deployed in molecular breeding for developing groundnut varieties with enhanced resistance to foliar diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9661-z) contains supplementary material, which is available to authorized users. 相似文献
889.
Robinson BG Khurana S Pohl JB Li WK Ghezzi A Cady AM Najjar K Hatch MM Shah RR Bhat A Hariri O Haroun KB Young MC Fife K Hooten J Tran T Goan D Desai F Husain F Godinez RM Sun JC Corpuz J Moran J Zhong AC Chen WY Atkinson NS 《PloS one》2012,7(5):e37394
Drosophila melanogaster has proven to be a useful model system for the genetic analysis of ethanol-associated behaviors. However, past studies have focused on the response of the adult fly to large, and often sedating, doses of ethanol. The pharmacological effects of low and moderate quantities of ethanol have remained understudied. In this study, we tested the acute effects of low doses of ethanol (~7 mM internal concentration) on Drosophila larvae. While ethanol did not affect locomotion or the response to an odorant, we observed that ethanol impaired associative olfactory learning when the heat shock unconditioned stimulus (US) intensity was low but not when the heat shock US intensity was high. We determined that the reduction in learning at low US intensity was not a result of ethanol anesthesia since ethanol-treated larvae responded to the heat shock in the same manner as untreated animals. Instead, low doses of ethanol likely impair the neuronal plasticity that underlies olfactory associative learning. This impairment in learning was reversible indicating that exposure to low doses of ethanol does not leave any long lasting behavioral or physiological effects. 相似文献
890.
K. Subrahmanya Prasad P. Biju K. Raveendran K. Gopalakrishna Bhat 《Nordic Journal of Botany》2012,30(1):58-60
A new aquatic species of the family Lythraceae (Rotala tulunadensis) collected from the lateritic plateau at Permude, Kerala, India is described and illustrated. It is closely allied to R. pterocalyx A. Raynal, but differs in having larger leaves, calyx tube not stretching laterally to include the capsule, calyx without interjected folds in fruit and larger petals. 相似文献