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Y C Liou

Publications and source records attributed to Y C Liou.

12 recordsLinked to original sources
PubMed2001

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins.

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.

Amino Acid Sequence↗
PubMed2001

Pin1 regulates turnover and subcellular localization of beta-catenin by inhibiting its interaction with APC.

Phosphorylation on a serine or threonine residue preceding proline (Ser/Thr-Pro) is a key regulatory mechanism, and the conformation of certain phosphorylated Ser/Thr-Pro bonds is regulated specifically by the prolyl isomerase Pin1. Whereas the inhibition of Pin1 induces apoptosis, Pin1 is strikingly overexpressed in a subset of human tumours. Here we show that Pin1 regulates beta-catenin turnover and subcellular localization by interfering with its interaction with adenomatous polyposis coli protein (APC). A differential-display screen reveals that Pin1 increases the transcription of several beta-catenin target genes, including those encoding cyclin D1 and c-Myc. Manipulation of Pin1 levels affects the stability of beta-catenin in vitro. Furthermore, beta-catenin levels are decreased in Pin1-deficient mice but are increased and correlated with Pin1 overexpression in human breast cancer. Pin1 directly binds a phosphorylated Ser-Pro motif next to the APC-binding site in beta-catenin, inhibits its interaction with APC and increases its translocation into the nucleus. Thus, Pin1 is a novel regulator of beta-catenin signalling and its overexpression might contribute to the upregulation of beta-catenin in tumours such as breast cancer, in which APC or beta-catenin mutations are not common.

Adenomatous Polyposis Coli↗
PubMed2000

Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein.

Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-A resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat beta-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section.

Animals↗
PubMed2000

Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria.

The hyperactive antifreeze protein from the beetle, Tenebrio molitor, is an 8.5-kDa, threonine-rich protein containing 16 Cys residues, all of which are involved in disulfide bonds. When produced by Escherichia coli, the protein accumulated in the supernatant in an inactive, unfolded state. Its correct folding required days or weeks of oxidation at 22 or 4 degrees C, respectively, and its purification included the removal of imperfectly folded forms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional (1)H and two-dimensional (1)H total correlation spectroscopy spectra were particularly helpful in establishing the characteristics of the fully folded antifreeze in comparison to less well-folded forms. The recombinant antifreeze had no free -SH groups and was rapidly and completely inactivated by 10 mM DTT. It had a thermal hysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of approximately 1.0 degrees C at 10-20 mg/ml. The circular dichroism spectra of the beetle antifreeze had a superficial resemblance to those of alpha-helical proteins, but deconvolution of the spectra indicated the absence of alpha-helix and the presence of beta-structure and coil. NMR analysis and secondary structure predictions agree with the CD data and are consistent with a beta-helix model proposed for the antifreeze on the basis of its 12-amino-acid repeating structure and presumptive disulfide bond arrangement.

Animals↗
PubMed2000

Crystallization and preliminary X-ray analysis of insect antifreeze protein from the beetle Tenebrio molitor.

Hyperactive antifreeze protein from the beetle Tenebrio molitor (TmAFP) was produced in Escherichia coli and purified by gel-permeation chromatography and HPLC. An iodinated derivative was prepared by incubating the 8.5 kDa TmAFP with N-iodosuccinimide. Native and iodinated TmAFP produced two different crystal forms when crystallized using the hanging-drop vapor-diffusion technique. Native crystals were rectangular plates that diffracted to approximately 2.5 A resolution. They were monoclinic and belonged to the space group P2(1), with unit-cell dimensions a = 38.4, b = 73.4, c = 59.3 A, beta = 97.0 degrees. Crystals of iodinated TmAFP formed elongated hexagons that allowed data to be collected to approximately 1.4 A. These crystals belonged to the space group P6(1) (or P6(5)), with unit-cell dimensions a = 73.85, b = 73.85, c = 53.15 A. There were two molecules per asymmetric unit, which corresponds to V(m) = 2.46 A(3) Da(-1) and 51% solvent content. A twofold non-crystallographic symmetry was evident from self-rotation calculations.

Animals↗
PubMed1999

A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands.

BACKGROUND: THP12 is an abundant and extraordinarily hydrophilic hemolymph protein from the mealworm Tenebrio molitor and belongs to a group of small insect proteins with four highly conserved cysteine residues. Despite their sequence homology to odorant-binding proteins and pheromone-binding proteins, the function of these proteins is unclear. RESULTS: The first three-dimensional structure of THP12 has been determined by multidimensional NMR spectroscopy. The protein has a nonbundle helical structure consisting of six alpha helices. The arrangement of the alpha helices has a 'baseball glove' shape. In addition to the hydrophobic core, electrostatic interactions make contributions to the overall stability of the protein. NMR binding studies demonstrated the binding of small hydrophobic ligands to the single hydrophobic groove in THP12. Comparing the structure of THP12 with the predicted secondary structure of homologs reveals a common fold for this new class of insect proteins. A search with the program DALI revealed extensive similarity between the three-dimensional structure of THP12 and the N-terminal domain (residues 1-95) of recoverin, a member of the family of calcium-binding EF-hand proteins. CONCLUSIONS: Although the biological function of this new class of proteins is as yet undetermined, a general role as alpha-helical carrier proteins for small hydrophobic ligands, such as fatty acids or pheromones, is proposed on the basis of NMR-shift perturbation spectroscopy.

Amino Acid Sequence↗
PubMed1999

A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor.

We have previously identified a Thr- and Cys-rich thermal hysteresis (antifreeze) protein (THP) in the beetle Tenebrio molitor that has 10-100 times the freezing point depression activity of fish antifreeze proteins. Because this 8.4 kDa protein is significantly different in its properties from THP preparations previously reported from this insect, a thorough search was undertaken for other antifreeze types. Many active proteins were observed, but all appeared to be isoforms of the THP that differed in their number of 12-amino acid repeats (consensus sequence CTxSxxCxxAxT), amino acid substitutions, and N-linked glycosylation. Mass spectral analysis has matched most of these isoforms with cDNA sequences of 17 different clones from a larval fat body library that encode eight different mature THPs containing 84, 96, or 120 amino acids. Genomic Southern blots suggest there may be 30-50 tightly linked copies of the gene, which is a signature consistently seen with unrelated fish antifreeze protein genes, and one that has been associated with the need to rapidly increase gene product in response to climate change. A three-dimensional model is proposed for the fully disulfide-bonded structure of T. molitor THP, which can accommodate addition or deletion of 12-amino acid repeats. The structure is a beta-helix that places most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.

Amino Acid Sequence↗
PubMed1997

Backbone structure and dynamics of a hemolymph protein from the mealworm beetle Tenebrio molitor.

Pheromones play a vital role in the survival of insects and are used for chemical communication between members of the same species by their olfactory system. The selection and transportation of these lipophilic messengers by carrier proteins through the hydrophilic sensillum lymph in the antennae toward their membrane receptors remains the initial step for the signal transduction pathway. A moderately abundant 12.4 kDa hydrophilic protein present in hemolymph from the mealworm beetle Tenebrio molitor is approximately 38% identical to a family of insect pheromone-binding proteins. The backbone structure and dynamics of the 108-residue protein have been characterized using three-dimensional 1H-15N NMR spectroscopy, combined with 15N relaxation and 1H/D exchange measurements. The secondary structure, derived from characteristic patterns of dipolar connectivities between backbone protons, secondary chemical shifts, and homonuclear three-bond JHNH alpha coupling constants, consists of a predominantly disordered N-terminus from residues 1 to 10 and six alpha-helices connected by four 4-7 residue loops and one beta-hairpin structure. The up-and-down arrangement of alpha-helices is stabilized by two disulfide bonds and hydrophobic interactions between amphipathic helices. The backbone dynamics were characterized by the overall correlation time, order parameters, and effective correlation times for internal motions. Overall, a good correlation between secondary structure and backbone dynamics was found. The 15N relaxation parameters T1 and T2 and steady-state NOE values of the six alpha-helices could satisfactorily fit the Lipari-Szabo model. In agreement with their generalized order parameters (> 0.88), residues in helical regions exhibited restricted motions on a picosecond time scale. The stability of this highly helical protein was confirmed by thermal denaturation studies.

Amides↗
PubMed1996

Malignant familial hypertrophic cardiomyopathy in a family with a 453Arg-->Cys mutation in the beta-myosin heavy chain gene: coexistence of sudden death and end-stage heart failure.

Recent genotype-phenotype correlation studies in familial hypertrophic cardiomyopathy (FHC) have revealed that some mutations in the beta- myosin heavy chain (BMHC) gene may be associated with a high incidence of sudden death and a poor prognosis. Coexistence of sudden death and end-stage heart failure in several families with FHC has recently being reported; however, the genetic basis of such families has not been clearly demonstrated. A three-generation Chinese familial hypertrophic cardiomyopathy (FHC) family (family HLI) with two cases of end-stage heart failure and three cases of sudden death was analyzed. The average age of death in the affected members in this family was 34 years old. Genetic linkage analysis using polymorphisms in the (alpha- and beta-myosin heavy chain genes revealed that FHC in this family is significantly linked to the BMHC gene without recombinations. Single-strand conformation polymorphism analysis of exons 8, 9 and 13 to 23 in the BMHC gene showed a polymorphic band on exon 14 that is in complete linkage with the disease status in this family. DNA sequencing analysis in the affected members revealed an 453Arg-->Cys mutation in the BMHC gene. To our knowledge this is the first reported mutation of FHC in Chinese. Our data suggest that the 453Arg-->Cys mutation is associated with a malignant clinical course in FHC due not only to sudden death but also to end-stage heart failure.

Adolescent↗