XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies.

IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.

Population-based carrier screening and prenatal diagnosis of fragile X syndrome in East Asian populations.

Identification of carriers of fragile X syndrome (FXS) with the subsequent prenatal diagnosis and knowledge of FXS-associated genetic profiles are essential for intervention in specific populations. We report the results of carrier screening of 39,458 East Asian adult women and prenatal diagnosis from 87 FXS carriers. The prevalence of FXS carriers and full mutation fetuses was estimated to be 1/581 and 1/3124 in East Asian populations, respectively. We confirmed the validity of the current threshold of CGG trinucleotide repeats for FMR1 categorization; the integral risks of full mutation expansion were approximately 6.0%, 43.8%, and 100% for premutation alleles with 55-74, 75-89, and ≥ 90 CGG repeats, respectively. The protective effect of AGG (adenine-guanine-guanine nucleotides) interruption in East Asian populations was validated, which is important in protecting premutation alleles with 75-89 CGG repeats from full mutation expansion. Finally, family history was shown not an effective indicator for FXS carrier screening in East Asian populations, and population-based screening was more cost-effective. This study provides an insight into the largest carrier screening and prenatal diagnosis for FXS in East Asian populations to date. The FXS-associated genetic profiles of East Asian populations are delineated, and population-based carrier screening is shown to be promising for FXS intervention.

Role of Pore-Lining Residues in Defining the Rate of Water Conduction by Aquaporin-0.

Compared to other aquaporins (AQPs), lens-specific AQP0 is a poor water channel, and its permeability was reported to be pH-dependent. To date, most water conduction studies on AQP0 were performed on protein expressed in Xenopus oocytes, and the results may therefore also reflect effects introduced by the oocytes themselves. Experiments with purified AQP0 reconstituted into liposomes are challenging because the water permeability of AQP0 is only slightly higher than that of pure lipid bilayers. By reconstituting high amounts of AQP0 and using high concentrations of cholesterol to reduce the permeability of the lipid bilayer, we improved the signal-to-noise ratio of water permeability measurements on AQP0 proteoliposomes. Our measurements show that mutation of two pore-lining tyrosine residues, Tyr-23 and Tyr-149 in sheep AQP0, to the corresponding residues in the high-permeability water channel AQP1 have additive effects and together increase the water permeability of AQP0 40-fold to a level comparable to that of AQP1. Molecular dynamics simulations qualitatively support these experimental findings and suggest that mutation of Tyr-23 changes the pore profile at the gate formed by residue Arg-187.

Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.

Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 Å crystal structure of the long-chain acyl-CoA carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent carboxylases. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.

Molecular architecture of the αß T cell receptor-CD3 complex.

αß T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR ß-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.

Structural study of the RIPoptosome core reveals a helical assembly for kinase recruitment.

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains.

Molecular mechanism for p202-mediated specific inhibition of AIM2 inflammasome activation.

Mouse p202 containing two hemopoietic expression, interferon inducibility, nuclear localization (HIN) domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found that only HIN1 of p202 binds double-stranded DNA (dsDNA), while HIN2 forms a homotetramer. Crystal structures of HIN1 revealed that dsDNA is bound on face opposite the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with the AIM HIN domain. We propose that this results in a spatial separation of the AIM2 pyrin domains, and indeed p202 prevented the dsDNA-dependent clustering of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility.

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of ß-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

Crystal structures of respiratory pathogen neuraminidases.

Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7A resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.

Colonization of a medical center in Southern Taiwan by epidemic strains of carbapenem- and multidrug-resistant Acinetobacter baumannii and the genetic organization of their integrons.

A total of 46 carbapenem- and multidrug-resistant (CR- and MDR-)Acinetobacter baumannii bacteremic isolates from a Taiwanese medical center were investigated over the period 2000 to 2006 using randomly amplified polymorphic DNA (RAPD) profiling and by analysing the genetic organization of their integrons. The results of RAPD patterns revealed that before 2003 each CR- and MDR-A. baumannii bacteremic isolate was independent, but after 2003 the isolates appeared to belong in four epidemic strains and persisted in the hospital. All the CR- and MDR-A. baumannii strains harbored class I integron (intI1) genes. PCR amplification and nucleotide sequencing showed that the cassette genes of intI1 were found to form four different antibiotic-resistant gene alignments in those strains. The bla(IMP-1) gene in the cassette genes of intI1 was identified in a clone, which raised great concern that clonal spread of this strain or of an integron-mediated horizontal gene may have occurred.

Phenotypic characteristics and pathogenic ability across distinct morphotypes of Burkholderia pseudomallei DT.

Burkholderia pseudomallei DT is unusual as it exhibits six distinct colony morphotypes. Types III and V show stronger motility, whereas type VI exhibits the highest levels of bacterial association with peritoneal exudate cells. Although the bacterial loads in the organs are not significantly different for infections by the six distinct morphotypes, higher mortality (100% and 89%, respectively) and larger areas of abnormal liver debris (20.6% and 22.4%, respectively) are found with types I- and III-infected mice compared to the others. These morphotypes sometimes undergo switching to a mucoid type in the body of mice, but the reverse has never been observed.

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.

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.

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.

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.

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.

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.

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.

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.

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.