Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

Interpreting Metabolomic Profiles using Unbiased Pathway Models

Human disease is heterogeneous, with similar disease phenotypes resulting from distinct combinations of genetic and environmental factors. Small-molecule profiling can address disease heterogeneity by evaluating the underlying biologic state of individuals through non-invasive interrogation of plasma metabolite levels. We analyzed metabolite profiles from an oral glucose tolerance test (OGTT) in 50 individuals, 25 with normal (NGT) and 25 with impaired glucose tolerance (IGT). Our focus was to elucidate underlying biologic processes. Although we initially found little overlap between changed metabolites and preconceived definitions of metabolic pathways, the use of unbiased network approaches identified significant concerted changes. Specifically, we derived a metabolic network with edges drawn between reactant and product nodes in individual reactions and between all substrates of individual enzymes and transporters. We searched for “active modules”—regions of the metabolic network enriched for changes in metabolite levels. Active modules identified relationships among changed metabolites and highlighted the importance of specific solute carriers in metabolite profiles. Furthermore, hierarchical clustering and principal component analysis demonstrated that changed metabolites in OGTT naturally grouped according to the activities of the System A and L amino acid transporters, the osmolyte carrier SLC6A12, and the mitochondrial aspartate-glutamate transporter SLC25A13. Comparison between NGT and IGT groups supported blunted glucose- and/or insulin-stimulated activities in the IGT group. Using unbiased pathway models, we offer evidence supporting the important role of solute carriers in the physiologic response to glucose challenge and conclude that carrier activities are reflected in individual metabolite profiles of perturbation experiments. Given the involvement of transporters in human disease, metabolite profiling may contribute to improved disease classification via the interrogation of specific transporter activities

Monitoring activity-dependent bulk endocytosis with the genetically-encoded reporter VAMP4-pHluorin

AbstractBackgroundActivity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during intense neuronal activity, implicating it as a major contributor to presynaptic plasticity under these stimulation conditions. However methods to monitor this endocytosis mode have been limited to either morphological or optical observation of the uptake of large fluid phase markers.New methodWe present here a method to monitor ADBE using the genetically-encoded reporter VAMP4-pHluorin in primary neuronal cultures.ResultsIndividual nerve terminals expressing VAMP4-pHluorin display either an increase or decrease in fluorescence after stimulation terminates. The decrease in fluorescence reflects the slow acidification of large bulk endosomes to which VAMP4-pHluorin is selectively recruited. Use of VAMP4-pHluorin during sequential high frequency stimuli revealed that all nerve terminals perform ADBE, but not all do so in response to a single stimulus. VAMP4-pHluorin also displays a rapid activity-dependent decrease in fluorescence during high frequency stimulation, a response which is particularly prominent when expressed in hippocampal neurons. The molecular mechanism responsible for this decrease is still unclear, but is not due to loss of VAMP4-pHluorin from the nerve terminal.Comparison with existing methodsThis method allows the selective reporting of ADBE for the first time, when compared to previous approaches using markers of fluid phase uptake.ConclusionsThe development of VAMP4-pHluorin as a selective genetically-encoded reporter of ADBE increases the palette of approaches used to monitor this endocytosis mode both in vitro and in vivo

Identification of novel functional sequence variants in the gene for peptidase inhibitor 3

BACKGROUND: Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied. METHODS: Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by χ(2 )goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm. RESULTS: Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF ≤ 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift. CONCLUSION: The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases

Proteomic analysis identifies proteins that continue to grow hepatic stem-like cells without differentiation

To understand the molecular mechanism underlying vigorous proliferative activity of hepatic stem-like (HSL) cells, we performed two-dimensional electrophoresis to identify the proteins statistically more abundant in rapidly growing undifferentiated HSL cells than in sodium butyrate-treated differentiated HSL cells. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Mascot search identified 6 proteins including prohibitin, vimentin, ezrin, annexin A3, acidic ribosomal phosphoprotein P0 and Grp75. Prohibitin and vimentin control the mitogen-activated protein (MAP) kinase pathway. Ezrin is phosphorylated by various protein-tyrosine kinases and modulates interactions between cytoskeletal and membrane proteins. Annexin A3 has a role in DNA synthesis. Acidic ribosomal phosphoprotein P0 and Grp75 play in protein synthesis. These results suggest that the proteins related to the MAP kinase cascade had some role in continuous proliferation of HSL cells without differentiation

Rule-based modelling provides an extendable framework for comparing candidate mechanisms underpinning clathrin polymerisation

Abstract Polymerisation of clathrin is a key process that underlies clathrin-mediated endocytosis. Clathrin-coated vesicles are responsible for cell internalization of external substances required for normal homeostasis and life –sustaining activity. There are several hypotheses describing formation of closed clathrin structures. According to one of the proposed mechanisms cage formation may start from a flat lattice buildup on the cellular membrane, which is later transformed into a curved structure. Creation of the curved surface requires rearrangement of the lattice, induced by additional molecular mechanisms. Different potential mechanisms require a modeling framework that can be easily modified to compare between them. We created an extendable rule-based model that describes polymerisation of clathrin molecules and various scenarios of cage formation. Using Global Sensitivity Analysis (GSA) we obtained parameter sets describing clathrin pentagon closure and the emergence/production and closure of large-size clathrin cages/vesicles. We were able to demonstrate that the model can reproduce budding of the clathrin cage from an initial flat array

Dynamics of Kv1 Channel Transport in Axons

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K+ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons

Functional mapping of brain synapses by the enriching activity-marker SynaptoZip

Ideally, elucidating the role of specific brain circuits in animal behavior would require the ability to measure activity at all involved synapses, possibly with unrestricted field of view, thus even at those boutons deeply located into the brain. Here, we introduce and validate an efficient scheme reporting synaptic vesicle cycling in vivo. This is based on SynaptoZip, a genetically encoded molecule deploying in the vesicular lumen a bait moiety designed to capture upon exocytosis a labeled alien peptide, Synbond. The resulting signal is cumulative and stores the number of cycling events occurring at individual synapses. Since this functional signal is enduring and measurable both online and ex post, SynaptoZip provides a unique method for the analysis of the history of synaptic activity in regions several millimeters below the brain surface. We show its broad applicability by reporting stimulus-evoked and spontaneous circuit activity in wide cortical fields, in anesthetized and freely moving animals

Altered synaptobrevin-II trafficking in neurons expressing a synaptophysin mutation associated with a severe neurodevelopmental disorder

textabstractFollowing exocytosis, synaptic vesicles (SVs) have to be reformed with the correct complement of proteins in the correct stoichiometry to ensure continued neurotransmission. Synaptophysin is a highly abundant, integral SV protein necessary for the efficient retrieval of the SV SNARE protein, synaptobrevin II (sybII). However the molecular mechanism underpinning synaptophysin-dependent sybII retrieval is still unclear. We recently identified a male patient with severe intellectual disability, hypotonia, epilepsy and callosal agenesis who has a point mutation in the juxtamembrane region of the fourth transmembrane domain of synaptophysin (T198I). This mutation had no effect on the activity-dependent retrieval of synaptophysin that was tagged with the genetically-encoded pH-sensitive reporter (pHluorin) in synaptophysin knockout hippocampal cultures. This suggested the mutant has no global effect on SV endocytosis, which was confirmed when retrieval of a different SV cargo (the glutamate transporter vGLUT1) was examined. However neurons expressing this T198I mutant did display impaired activity-dependent sybII retrieval, similar to that observed in synaptophysin knockout neurons. Interestingly this impairment did not result in an increased stranding of sybII at the plasma membrane. Screening of known human synaptophysin mutations revealed a similar presynaptic phenotype between T198I and a mutation found in X-linked intellectual disability. Thus this novel human synaptophysin mutation has revealed that aberrant retrieval and increased plasma membrane localisation of SV cargo can be decoupled in human disease