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991.
Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for understanding their evolution and function but is not trivial. More specifically, partitioning domains that interact by forming a single β-sheet is known to be particularly troublesome for automatic structure-based domain decomposition pipelines. Here, we study edge-to-edge β-strand interactions between domains in a protein chain, to help define the boundaries for some more difficult cases where a single β-sheet spanning over two domains gives an appearance of one. We give a number of examples where β-strands belonging to a single β-sheet do not belong to a single domain and highlight the difficulties of automatic domain parsers on these examples. This work can be used as a baseline for defining domain boundaries in homologous proteins or proteins with similar domain interactions in the future. 相似文献
992.
Adaptive responses are induced by stress such as X radiation and result in a lower than expected biological response. Two-dose adaptive response experiments typically involve a low priming dose followed by a subsequent high radiation dose. Here, we used a sensitive in vivo chromosomal inversion assay to demonstrate for the first time an adaptive response when a low dose (0.01-1 mGy) was given several hours after a high 1000-mGy radiation dose. The adaptive responses in this study were of similar magnitude to the two-dose adaptive responses previously observed in this test system when the low dose was given first. A chromosomal inversion adaptive response was also induced by two 1000-mGy doses and when a 1-mGy dose was preceded or followed by a dose of 0.01 mGy, but not by two 4000-mGy doses. This is also the first example of an adaptive response when both doses are low. Our data agree with previous reports of an on-off mechanism of adaptive response. The induction of an adaptive response by a low dose after a high damaging dose provides evidence that the mechanisms underlying radiation adaptive responses are not due to prevention of damage induced by the high dose but to modulation of the cellular response to this damage. 相似文献
993.
Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site. 相似文献
994.
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
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998.
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
999.
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
1000.
Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A. 相似文献