Biomedical subjects

Yu-Shan Hsiao

Publications and source records attributed to Yu-Shan Hsiao.

9 recordsLinked to original sources

PubMed2006

Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates.

Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

Acetyl Coenzyme A↗

PubMed2006

Crystal structure of rat carnitine palmitoyltransferase II (CPT-II).

Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the beta-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9A resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.

Amino Acid Sequence↗

PubMed2006

CpG-modified plasmid DNA encoding flagellin improves immunogenicity and provides protection against Burkholderia pseudomallei infection in BALB/c mice.

The plasmid DNA encoding the fliC gene of Burkholderia pseudomallei combined with CpG oligodeoxynucleotide (ODN) was injected intramuscularly into BALB/c mice, resulting in the increased production of certain humoral antibodies and flagellin-specific spleen cell clonal expansion. CpG ODN, as an immunoadjuvant, was added to the plasmid containing the fliC gene in order to obtain ongoing expression in muscle for a long period. Functional expression of flagellin from the constructed CpG-modified plasmid in transfected peritoneal exudate cells of BALB/c mice was shown by reverse transcription-PCR and Western blotting. Furthermore, BALB/c mice immunized with the modified plasmid had relatively higher resistance to B. pseudomallei infection in vivo than did mice immunized with unmodified plasmid DNA. The time course of restricted bacterial growth in spleen and liver and changes in the cytokine profiles of immunized mice suggested that the stimulated phagocytic cells would be able to kill the bacteria eventually, possibly as a consequence of the induction of Th-1-type immune polarization in vivo. Th-1-type immune polarization was detected in response to flagellin induction in mice immunized with CpG-modified plasmid DNA by the appearance of increased levels of immunoglobulin G2a antibodies and gamma interferon-secreting cells specific to flagellin. The exogenous CpG motifs added to the fliC gene would contribute to an adjuvant-like response that enhances the flagellin-specific immunogenicity and provides protection against B. pseudomallei infection. This CpG-modified plasmid DNA vaccination is an important potential strategy that should be developed to protect against melioidosis.

Adjuvants, Immunologic↗

PubMed2006

Bacterial neuraminidase facilitates mucosal infection by participating in biofilm production.

Many respiratory pathogens, including Hemophilus influenzae, Streptococcus pneumoniae, and Pseudomonas aeruginosa, express neuraminidases that can cleave alpha2,3-linked sialic acids from glycoconjugates. As mucosal surfaces are heavily sialylated, neuraminidases have been thought to modify epithelial cells by exposing potential bacterial receptors. However, in contrast to neuraminidase produced by the influenza virus, a role for bacterial neuraminidase in pathogenesis has not yet been clearly established. We constructed a mutant of P. aeruginosa PAO1 by deleting the PA2794 neuraminidase locus (Delta2794) and tested its virulence and immunostimulatory capabilities in a mouse model of infection. Although fully virulent when introduced i.p., the Delta2794 mutant was unable to establish respiratory infection by i.n. inoculation. The inability to colonize the respiratory tract correlated with diminished production of biofilm, as assessed by scanning electron microscopy and in vitro assays. The importance of neuraminidase in biofilm production was further demonstrated by showing that viral neuraminidase inhibitors in clinical use blocked P. aeruginosa biofilm production in vitro as well. The P. aeruginosa neuraminidase has a key role in the initial stages of pulmonary infection by targeting bacterial glycoconjugates and contributing to the formation of biofilm. Inhibiting bacterial neuraminidases could provide a novel mechanism to prevent bacterial pneumonia.

Amino Acid Sequence↗

PubMed2005

Immunogenicity and anti-Burkholderia pseudomallei activity in Balb/c mice immunized with plasmid DNA encoding flagellin.

Plasmid DNA encoding the flagella protein (flagellin) was used as a vaccination candidate for the evaluation of its immunogenicity and for protection against infection with Burkholderia pseudomallei. Firstly, flagellin encoding plasmid DNA was injected into Balb/c mice intramuscularly and this elicited both a humoral and a cellular immune response. Total IgG production and the clonal expansion of the spleen cells increased in response to flagellin. The IgG subclass response exhibited a dominance of IgG2a over IgG1 in the sera. In addition, IFN-gamma-secreting cells in the spleen were substantially increased. Furthermore, the anti-B. pseudomallei activity of the peritoneal exudate cells was evaluated by a Transwell tissue-culture plate system where the macrophage-activating related cytokines in upper chamber were allowed to cross the plate's membrane and stimulate the activation of peritoneal exudate cells in lower chamber. Our results indicated that the activated peritoneal exudate cells were able to restrict the growth of B. pseudomallei in vitro. Indeed, subsequent intravenous challenge of the vaccinated Balb/c mice with 10(5)CFU of B. pseudomallei resulted in the number of bacterial cells detected in liver and/or spleen being significantly reduced in the flagellin plasmid DNA vaccinated mice. At 7 days subsequent to infection of B. pseudomallei, 5/6 (83%) of flagellin plasmid DNA vaccinated mice had survived. We suggest that plasmid DNA-encoding flagellin might be useful as a potential immunization route for the future development of a vaccine against melioidosis in related animals.

Animals↗

PubMed2004

Crystal structure of mouse carnitine octanoyltransferase and molecular determinants of substrate selectivity.

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.

Amino Acid Sequence↗

PubMed2004

Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase.

Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme.

Acyl Coenzyme A↗

PubMed2004

Structure and function of carnitine acyltransferases.

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and coenzyme A (CoA). These enzymes include carnitine acetyltransferase (CrAT), carnitine octanoyltransferase (CrOT), and carnitine palmitoyltransferases (CPTs). CPT-I and CPT-II are crucial for the beta-oxidation of long-chain fatty acids in the mitochondria by enabling their transport across the mitochondrial membrane. The activity of CPT-I is inhibited by malonyl-CoA, a crucial regulatory mechanism for fatty acid oxidation. Mutation or dysregulation of the CPT enzymes has been linked to many serious, even fatal human diseases, and these enzymes are promising targets for the development of therapeutic agents against type 2 diabetes and obesity. We have determined the crystal structures of murine CrAT, alone and in complex with its substrate carnitine or CoA. The structure contains two domains. Surprisingly, these two domains share the same backbone fold, which is also similar to that of chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. The active site is located at the interface between the two domains, in a tunnel that extends through the center of the enzyme. Carnitine and CoA are bound in this tunnel, on opposite sides of the catalytic His343 residue. The structural information provides a molecular basis for understanding the catalysis by carnitine acyltransferases and for designing their inhibitors. In addition, our structural information suggests that the substrate carnitine may assist the catalysis by stabilizing the oxyanion in the reaction intermediate.

Acetyltransferases↗

PubMed2002

A single mutation converts the nucleotide specificity of phenol sulfotransferase from PAP to AMP.

Sulfotransferases (STs) catalyze all the known biological sulfonations, in which a sulfuryl group from a common sulfonate donor such as 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is transferred to a nucleophilic acceptor. In addition to PAPS, phenol sulfotransferase (PST), a member of the ST family, utilizes other nucleotides as substrates with much less catalytic efficiency [Lin, E. S., and Yang, Y. S. (2000) Biochem. Biophys. Res. Commun. 271, 818-822]. Six amino acid residues of PST have been chosen for mutagenesis studies on the basis of a model of PST and its sequence alignment with those of available cytosolic and membrane-anchored STs. Systematic analyses of the mutants reveal that Ser134 is important for the regulation of nucleotide specificity between 3'-phosphoadenosine 5'-phosphate (PAP) and adenosine 5'-monophosphate (AMP). Kinetic studies also indicate that Ser134 plays a key role in nucleotide binding (K(m)) but not in catalysis (kcat). Consequently, the catalytic efficiency (kcat/K(m)) of PST can be altered by 5 orders of magnitude with a mutation of Ser134. Moreover, the change in nucleotide specificity from PAP to AMP can be achieved by mutation of Ser134 to any of the following residues: Glu, Gln, Arg, and His. Roles of Lys44, Arg126, and Arg253, which interact directly with the 5'- and 3'-phosphate of PAP, were also investigated by mutagenesis and kinetic experiments. On the basis of these findings, we suggest that Ser134 is the key residue that enables PST to discriminate PAP from AMP.

Adenosine Diphosphate↗