Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria

Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories:'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.

Rapid radiation-induction of atm protein levels in situ

Ataxia-telangiectasia (A-T) is characterised by hypersensitivity to ionising radiation (IR), immunodeficiency, neurodegeneration and predisposition to malignancy. Mutations in the A-T gene (ATM) often result in reduced levels of ATM protein and/or compromise ATM function. IR induced DNA damage is known to rapidly upregulate ATM kinase activity/phosphorylation events in the control of cell cycle progression and other processes. Variable expression of ATM levels in different tissues and its upregulation during cellular proliferation indicate that the level of ATM is also regulated by mechanisms other than gene mutation. Here, we report on the IR induction of ATM protein levels within a number of different cell types and tissues. Induction had begun within 5 min and peaked within 2 h of exposure to 2 Gy of IR, suggesting a rapid post-translational mechanism. Low basal levels of ATM protein were more responsive to IR induction compared to high ATM levels in the same cell type. Irradiation of fresh skin biopsies led to an average three-fold increase in ATM levels while immunohistochemical analyses indicated low expressing cells within the basal layer with ten-fold increases in ATM levels following IR. ATM high expressing lymphoblastoid cell lines (LCLs) which were initially resistant to the radiation-induction of ATM levels also became responsive to IR after ATM antisense expression was used to reduce the basal levels of the protein. These results demonstrate that ATM is present in variable amounts in different tissue/cell types and where basal levels are low ATM levels can be rapidly induced by IR to saturable levels specific for different cell types. ATM radiation-induction is a sensitive and rapid radioprotective response that complements the IR mediated activation of ATM.

Ataxia-telangiectasia: chronic activation of damage-responsive functions is reduced by alpha-lipoic acid

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress, Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha -lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

Intrinsic regional differences in androgen receptors and dihydrotestosterone metabolism in human preadipocytes

Androgens play an important role in regulating the central obesity that is a strong risk factor for cardiovascular disease and insulin resistance. This study confirms that androgen receptors are present in subcultured human preadipocytes, with androgen receptor gene expression and saturable specific dihydrotestosterone binding, dissociation constant 1.02 - 2.56 nM and maximal binding capacity 30.8 - 55.7 fmol/mg protein. There was an intrinsic regional difference in androgen receptor complement, with more androgen receptors in visceral than in subcutaneous preadipocytes. Dihydrotestosterone was metabolised by human preadipocytes, with more androstanediol produced by subcutaneous than visceral preadipocytes. While dihydrotestosterone metabolism was insufficient to explain the regional variation in androgen binding, both of these differences would reduce the androgen responsiveness of the subcutaneous preadipocytes compared with visceral preadipocytes. There were no gender differences in androgen binding or metabolism. While the direct effects of androgens on human PAS remain uncertain, these regional differences suggest that AR-mediated regulation of certain PA functions influences adipose tissue distribution.

Storage protein mobilization and thiol protease up-regulation by solid matrix priming in loblolly pine (Pinus taeda) seed embryos

Experiments were conducted to investigate physiological mechanisms of solid matrix priming (SMP) on germination enhancement of loblolly pine (Pinus taeda) seeds. During SMP, osmotic potential in the embryo decreased by 0.65 MPa, concentration of crystalloid proteins decreased to 62% and concentrations of buffer soluble proteins and free amino acids increased by 22% and by 166%, respectively. Observations under an electron microscope demonstrated protein bodies in the embryo were mobilized. Inhibitor analysis indicated thiol protease was the dominant enzyme among endopiptidases to degrade the reserved proteins. A fragment of thiol protease was cloned from the primed seed embryos and it has high identities to those thiol proteases responsive to water stress. RNA get blot analysis showed a 1.5 kb thiol protease gene was up-regulated by SMP. Treatment with E64, a thiol protease inhibitor, negated SMP effects on germination performance, water potentials and protein profiles. Based on the experimental results, reserve protein mobilization induced by SMP in the embryo before radicle emergence might be one of the mechanisms to enhance germination in loblolly pine seeds.

Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts

The addition of insulin during in vitro culture has beneficial effects on rabbit preimplantation embryos leading to increased cell proliferation and reduced apoptosis. We have previously described the expression of the insulin receptor (IR) and the insulin-responsive glucose transporters (GLUT) 4 and 8 in rabbit preimplantation embryos. However, the effects of insulin on IR signaling and glucose metabolism have not been investigated in rabbit embryos. In the present study, the effects of 170 nM insulin on IR, GLUT4 and GLUT8 mRNA levels, Akt and Erk phosphorylation, GLUT4 translocation and methyl glucose transport were studied in cultured day 3 to day 6 rabbit embryos. Insulin stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) Erk1/2 and levels of IR and GLUT4 mRNA, but not phosphorylation of the phosphatidylinositol 3-kinase-dependent protein kinase, Akt, GLUT8 mRNA levels, glucose uptake or GLUT4 translocation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control.

GLUT4 overexpression or deficiency in adipocytes of transgenic mice alters the composition of GLUT4 vesicles and the subcellular localization of GLUT4 and insulin-responsive aminopeptidase

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2GLUT4- Tg) mice were studied. GLUT4 amount was reduced by 80 - 95% in aP2-GLUT4-/- adipocytes and increased similar to10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase ( IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2GLUT4- Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.

Selenium binding protein 1 in ovarian cancer

Selenium binding protein I (SELENBP1) was identified to be the most significantly down-regulated protein in ovarian cancer cells by a membrane proteome profiling analysis. SELENBP1 expression levels in 4 normal ovaries, 8 benign ovarian tumors, 12 borderline ovarian tumors and 141 invasive ovarian cancers were analyzed with immunohistochemical assay. SELENBP1 expression was reduced in 87% cases of invasive ovarian cancer (122/141) and was significantly reduced in borderline tumors and invasive cancers (p < 0.001). Cox multivariate analysis within the 141 invasive cancer tissues showed that SELENBP1 expression score was a potential prognostic indicator for unfavorable prognosis of ovarian cancer (hazard ratio [HR], 2.18; 95% CI = L22-190; p = 0.009). Selenium can disrupt the androgen pathway, which has been implicated in modulating SELENBP1 expression. We investigated the effects of selenium and androgen on normal human ovarian surrace epithelial (HOSE) cells and cancer cells. Interestingly, SELENBP1 mRNA and protein levels were reduced by androgen and elevated by selenium treatment in the normal HOSE cells, whereas reversed responses were observed in the ovarian cancer cell lines. These results suggest that changes of SELENBP1 expression in malignant ovarian cancer are an indicator of aberration of selenium/androgen pathways and may reveal prognostic information of ovarian cancer. (c) 2005 Wiley-Liss, Inc.

Foaming properties of a peptide designed to form stimuli-responsive interfacial films

We have designed an amphipathic peptide, AM1, that can self-assemble at the air-water interface to form an interfacial ensemble capable of switching between a mechanically strong cohesive film state and a mobile detergent state in response to changes in the solution conditions. The mechanical properties of the AM1 ensemble in the cohesive film state are qualitatively equivalent to the protein beta-LG, while in the mobile detergent state they are equivalent to the low molecular weight surfactant, SDS. In this work the foaming properties of AM1 are compared to those of beta-LG and SDS at the same weight concentration and it is found that AM1 adsorbs rapidly to the interface, initially forming a dense foam like that formed by SDS and superior to beta-LG. In addition, under solution conditions where interfacially adsorbed AM1 forms a cohesive film state the foam stability is high, comparable to beta-LG. However when the interfacially adsorbed AM1 forms a foam under detergent-state conditions, the foam stability is poor. We have achieved control of foam stability through the design of a peptide that exhibits stimuli-responsive changes in the extent of intermolecular interactions between peptide molecules adsorbed at the air water interface. These results illustrate the exciting potential of peptide surfactants to form a new class of stimuli-responsive foaming agents.

Cross-sectional and prospective relationships of interleukin-6 and C-reactive protein with physical performance in elderly persons: MacArthur Studies of Successful aging

Although IL-6 has been shown to predict onset of disability in older persons and both IL-6 and CRP are associated with motality risk, these markers of inflammation have only limited associations with physical performance, except for walking measures and grip strength at baseline, and do not predict change in performance 7 years later in a high-functioning subset of older adults.

Geographic variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement, which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of reproductive character displacement; there are no unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Analysis of Wolbachia protein synthesis in Drosophila in vivo

Intracellular Wolbachia infections are extremely common in arthropods and exert profound control over the reproductive biology of the host. However, very little is known about the underlying molecular mechanisms which mediate these interactions with the host. We examined protein synthesis by Wolbachia in a Drosophila host in vivo by selective metabolic labelling of prokaryotic proteins and subsequent analysis by 1D and 2D gel electrophoresis. Using this method we could identify the major proteins synthesized by Wolbachia in ovaries and testes of flies. Of these proteins the most abundant was of low molecular weight and showed size variation between Wolbachia strains which correlated with the reproductive phenotype they generated in flies. Using the gel systems we employed it was not possible to identify any proteins of Wolbachia origin in the mature sperm cells of infected flies.

Positive selection on an acrosomal sperm protein, M7 lysin, in three species of the mussel genus Mytilus

Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of inter and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and non-synonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, shows that positive selection on sperm proteins can occur even when post-zygotic reproductive isolation is incomplete.

Sequence and tissue distribution of chicken cellular nucleic acid binding protein cDNA

We sequenced cDNAs coding for chicken cellular nucleic acid binding protein (CNBP). Two slightly different variations of the open reading frame were found, each of which translates into a protein with seven zinc finger domains. The longest transcript contains an in-frame insert of 3 bp. The sequence conservation between chick CNBP cDNAs with human, rat and mouse CNBP cDNAs is extreme, especially in the coding region, where the deduced amino acid sequence identity with human, rat and mouse CNBP is 99%. CNBP-like transcripts were also found in various tissues from insect, shrimp, fish and lizard. Regions with remarkable nucleotide conservation were also found in the 3' untranslated region, indicating important functions for these regions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that in the chick, CNBP is present in all tissues examined in approximately equal ratios to total RNA. RT-PCR of total RNA isolated from different phyla indicate CNBP-like proteins art widespread throughout the animal kingdom. The extraordinary level of conservation suggests an important physiological role for CNBP. (C) 1997 Elsevier Science Inc.