The VAP protein family: from cellular functions to motor neuron disease

The VAMP-associated proteins (VAPs) are highly conserved integral endoplasmic reticulum membrane proteins implicated in diverse cellular functions, including the regulation of lipid transport and homeostasis, membrane trafficking, neurotransmitter release, stabilization of presynaptic microtubules, and the unfolded protein response. Recently, a single missense mutation within the human VAP-B gene was identified in three forms of familial motor neuron disease. In this review, we integrate results from studies of yeast, fly and mammalian VAPs that provide insight into the structural features of these proteins, the network of VAP-interacting proteins, their possible physiological functions, and their involvement in motor neuron disease.     

PCRless library mutagenesis via oligonucleotide recombination in yeast

The directed evolution of biomolecules with new functions is largely performed in vitro, with PCR mutagenesis followed by high-throughput assays for desired activities. As synthetic biology creates impetus for generating biomolecules that function in living cells, new technologies are needed for performing mutagenesis and selection for directed evolution in vivo. Homologous recombination, routinely exploited for targeted gene alteration, is an attractive tool for in vivo library mutagenesis, yet surprisingly is not routinely used for this purpose. Here, we report the design and characterization of a yeast-based system for library mutagenesis of protein loops via oligonucleotide recombination. In this system, a linear vector is co-transformed with single-stranded mutagenic oligonucleotides. Using repair of nonsense codons engineered in three different active-site loops in the selectable marker TRP1 as a model system, we first optimized the recombination efficiency. Single-loop recombination was highly efficient, averaging 5%, or 4.0×10(5) recombinants. Multiple loops could be simultaneously mutagenized, although the efficiencies dropped to 0.2%, or 6.0×10(3) recombinants, for two loops and 0.01% efficiency, or 1.5×10(2) recombinants, for three loops. Finally, the utility of this system for directed evolution was tested explicitly by selecting functional variants from a mock library of 1:10(6) wild-type:nonsense codons. Sequencing showed that oligonucleotide recombination readily covered this large library, mutating not only the target codon but also encoded silent mutations on either side of the library cassette. Together these results establish oligonucleotide recombination as a simple and powerful library mutagenesis technique and advance efforts to engineer the cell for fully in vivo directed evolution.     

Differential expression of GABA<Subscript>A</Subscript> receptor π subunit in cultured rat alveolar epithelial cells

Although type A -aminobutyric acid (GABA) receptors (ligand-gated Cl^– channels) have been extensively studied in the central nervous system, no information is available on this receptor in lung cells. We have examined the expression of GABA_A receptor -subunit (GABRP) during the trans-differentiation between rat alveolar epithelial type II cells and type I cells. Rat alveolar type II cells, when cultured on plastic plates, gradually trans-differentiated into type-I-like cells and lost their GABRP mRNA expression. However, the GABRP mRNA was partially retained in the type II cells cultured on Matrigel. Keratinocyte growth factor (a mitogen of type II cells) increased GABRP expression. A detached collagen gel maintained the GABRP mRNA to a level close to that of the freshly isolated type II cells. An air–liquid interface culture system, mimicking in vivo conditions in the lung, significantly up-regulated the expression of GABRP mRNA and protein. mRNAs of the GABA_A receptor 1-, 3-, 2-, 2-, and 3-subunits were also detected in rat type II cells. These results suggest that GABRP expression is differentially regulated by culture substrata, growth factor, detached gel, and an air-apical surface.This work was supported by NIH R01 HL-52146, R01 NIH-071628, and OCAST HR01-093, and AHA heartland affiliate 0255992Z (to L.L.). N.J. was supported by an AHA heartland affiliate pre-doctoral fellowship (0315256Z).     

Correlations between the fMRI BOLD signal and visual perception

Using fMRI and a psychophysical task involving letter identification, Kleinschmidt et al. (2002) (this issue of Neuron) delineate two patterns of neural activation, which manifest in different cortical regions: a transient activation, correlated with the change of a percept, and a longer-term hysteresis, correlated with the maintenance of the percept. These findings are provocative and suggest that neural hysteresis is mediated by visual structures that interact with higher-order regions to support longer-term maintenance of a percept.     

Evidence for the interference of aluminum with bacterial porphyrin biosynthesis

Aluminum (0.74 mm) was found to retard bacterial growth, and enhance porphyrin formation and excretion in Arthrobacter aurescens RS-2. Coproporphyrin III was shown to be the main porphyrin excreted by aluminum-exposed A. aurescens RS-2 cultures and by RS-2 cultures grown under anoxic conditions. Synthesis and excretion of porphyrins in A. aurescens RS-2 increased in a dose-dependent manner when the bacteria were exposed to increasing aluminum concentrations. Incubation of A. aurescens RS-2 with -aminolevulinic acid (-ALA, 1.2 mm) brought about the intense formation and excretion of porphyrins by the cells, in the presence or absence of aluminum. -ALA slightly enhanced the toxicity of aluminum towards RS-2 bacteria. Furthermore, the intracellular concentration of heme was reduced by 63.9 ± 8.67% in aluminum-exposed RS-2 bacteria when compared with control cultures. The results are discussed in light of the recent finding concerning aluminum toxicity and porphyrin biosynthesis in microorganisms.     

Mutations in olfactory signal transduction genes are not a major cause of human congenital general anosmia

Anosmia affects the western world population, mostly the elderly, reaching to 5% in subjects over the age of 45 years and strongly lowering their quality of life. A smaller minority (about 0.01%) is born without a sense of smell, afflicted with congenital general anosmia (CGA). No causative genes for human CGA have been identified yet, except for some syndromic cases such as Kallman syndrome. In mice, however, deletion of any of the 3 main olfactory transduction components (guanidine triphosphate binding protein, adenylyl cyclase, and the cyclic adenosine monophosphate-gated channel) causes profound reduction of physiological responses to odorants. In an attempt to identify human CGA-related mutations, we performed whole-genome linkage analysis in affected families, but no significant linkage signals were observed, probably due to the small size of families analyzed. We further carried out direct mutation screening in the 3 main olfactory transduction genes in 64 unrelated anosmic individuals. No potentially causative mutations were identified, indicating that transduction gene variations underlie human CGA rarely and that mutations in other genes have to be identified. The screened genes were found to be under purifying selection, suggesting that they play a crucial functional role not only in olfaction but also potentially in additional pathways.     

Co-Sensitization to Silkworm Moth (Bombyx mori) and 9 Inhalant Allergens among Allergic Patients in Guangzhou,Southern China

Objectives

This study aimed to investigate the profile of sensitization to silkworm moth (Bombyx mori) and other 9 common inhalant allergens among patients with allergic diseases in southern China.

Methods

A total of 175 patients were tested for serum sIgE against silkworm moth in addition to combinations of other allergens: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Blattella germanica, Periplaneta americana, cat dander, dog dander, Aspergillus fumigatus and Artemisia vulgaris by using the ImmunoCAP system. Correlation between sensitization to silkworm moth and to the other allergens was analyzed.

Results

Of the 175 serum samples tested, 86 (49.14%) were positive for silkworm moth sIgE. With high concordance rates, these silkworm moth sensitized patients were concomitantly sensitized to Dermatophagoides pteronyssinus (94.34%), Dermatophagoides farinae (86.57%), Blomia tropicalis (93.33%), Blattella germanica (96.08%), and Periplaneta americana (79.41%). Moreover, there was a correlation in serum sIgE level between silkworm moth and Dermatophagoides pteronyssinus (r = 0.518), Dermatophagoides farinae (r = 0.702), Blomia tropicalis (r = 0.701), Blattella germanica (r = 0.878), and Periplaneta americana (r = 0.531) among patients co-sensitized to silkworm moth and each of these five allergens.

Conclusion

In southern Chinese patients with allergic diseases, we showed a high prevalence of sensitization to silkworm moth, and a co-sensitization between silkworm moth and other five common inhalant allergens. Further serum inhibition studies are warranted to verify whether cross-reactivity exists among these allergens.     

Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation

The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.     

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.