Biology subjects

Qiu, D.

Publications and source records attributed to Qiu, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Pools of independently cycling inositol phosphates revealed by pulse labeling with 18O-water

Inositol phosphates control many central processes in eukaryotic cells, including nutrient availability, growth, and motility. Kinetic resolution of a key modulator of their signaling functions, the turnover of the phosphate groups on the inositol ring, has been hampered by slow uptake, high dilution, and constraining growth conditions in radioactive pulse-labeling approaches. Here, we demonstrate rapid (seconds to minutes), non-radioactive labeling of inositol polyphosphates through 18O-water in yeast, amoeba and human cells, which can be applied in any media. In combination with capillary electrophoresis and mass spectrometry, 18O-water labeling simultaneously dissects the in vivo phosphate group dynamics of a broad spectrum of even rare inositol phosphates. The improved temporal resolution allowed us to discover vigorous phosphate group exchanges in some inositol poly- and pyrophosphates, whereas others remain remarkably inert. Our observations support a model in which the biosynthetic pathway of inositol poly- and pyrophosphates is organized in distinct, kinetically separated pools. While transfer of compounds between those pools is slow, each pool undergoes rapid internal phosphate cycling. This might enable the pools to perform distinct signaling functions while being metabolically connected.

biochemistry↗

bioRxiv2024

A novel classification framework for genome-wide association study of whole brain MRI images using deep learning

Genome-wide association studies (GWASs) have been widely applied in the neuroimaging field to discover genetic variants associated with brain-related traits. So far, almost all GWASs conducted in neuroimaging genetics are performed on univariate quantitative features summarized from brain images. On the other hand, powerful deep learning technologies have dramatically improved our ability to classify images. In this study, we proposed and implemented a novel machine learning strategy for systematically identifying genetic variants that lead to detectable nuances on Magnetic Resonance Images (MRI). For a specific single nucleotide polymorphism (SNP), if MRI images labeled by genotypes of this SNP can be reliably distinguished using machine learning, we then hypothesized that this SNP is likely to be associated with brain anatomy or function which is manifested in MRI brain images. We applied this strategy to a catalog of MRI image and genotype data collected by the Alzheimers Disease Neuroimaging Initiative (ADNI) consortium. From the results, we identified novel variants that show strong association to brain phenotypes.

bioinformatics↗

bioRxiv2024

In vitro determination of osteo-adipogenic lineage choice of bone marrow stromal/stem cells (BMSCs)

Bone marrow stromal/stem cells (BMSCs) are primitive and heterogeneous cells that can be differentiated into osteoblasts, adipocytes and other subsets. Their bone-fat lineage commitment is responsible for the homeostasis of bone marrow microenvironment. However, there are little effective methods and evidence to simultaneously evaluate the lineage commitment of BMSCs. Here we provide a mixed osteo-adipogenic (OA) differentiation medium that could enable BMSCs differentiation into osteoblasts and adipocytes in vitro, and establish a method for simultaneous assessment of bone-fat lineage allocation of BMSCs based on the Alizarin red S and Oil red O staining, which have been used for detection of specific mineralized nodules and lipid droplets, respectively. This assay provides a specifically simple but effective and low-cost method to visualize the osteogenesis and adipogenesis of BMSCs, and evaluate the differentiation efficiency of lineage allocation of BMSCs.

cell biology↗

bioRxiv2023

A 3-component module maintains sepal flatness in Arabidopsis

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding, through the interplay between biochemical and biomechanical cues. In contrast, certain organs maintain their flat posture over several days. Here we identified a pathway, which is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular and mechanical approaches, our results demonstrate that global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin signaling to down-regulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Our work unravels a 3-component module, which relates hormonal patterns to organ curvature, and actively maintains sepal flatness during its growth.

plant biology↗

bioRxiv2023

Conservation of heat stress acclimation by the inositol polyphosphate multikinase, IPMK responsible for 4/6-InsP7 production in land plants

Inositol pyrophosphates (PP-InsPs) are soluble cellular messengers that integrate environmental cues to induce adaptive responses in eukaryotes. In plants, the biological functions of various PP-InsP species are poorly understood, largely due to the absence of canonical enzymes present in other eukaryotes. The recent identification of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP7 in the eudicot Arabidopsis thaliana, further highlights the intricate PP-InsP signalling network employed by plants. The abundance of 4/6-InsP7 in land plants, the enzyme(s) responsible for its synthesis, and the physiological functions of this species are all currently unknown. In this study, we show that 4/6-InsP7 is the major PP-InsP species present across land plants. Our findings demonstrate that the Arabidopsis inositol polyphosphate multikinase (IPMK) homolog, AtIPK2 generates 4/6-InsP7 in vitro. Furthermore, the cellular level of 4/6-InsP7 is controlled by the two Arabidopsis IPMK isoforms, AtIPK2 and AtIPK2{beta}. Notably, the activity of these IPMK proteins is critical for heat stress acclimation in Arabidopsis. During heat stress, the expression of genes encoding various heat shock proteins controlled by the heat shock factors (HSFs) is affected in the AtIPK2-deficient plants. Furthermore, we show that the transcription activity of HSF is regulated by the AtIPK2 proteins. Our parallel investigations using the liverwort Marchantia polymorpha suggest that the InsP6 kinase activity of IPMK and the role of IPMK in regulating the heat stress response are evolutionarily conserved. Collectively, our study indicates that IPMK has played a critical role in transducing environmental cues for biological processes during land plant evolution.

plant biology↗

bioRxiv2023

The impact of multiband and in-plane acceleration on white matter microstructure analysis

Accelerated imaging has been broadly adopted in diffusion MRI studies, yet little is known about its impacts. Acceleration can achieve higher spatial and q-space resolution in shorter time, reduce motion artifacts, and reduce patient burden. However, it leads to noise amplification, and its impacts in clinical cohorts are poorly understood. This study examined the impact of multiband (also called simultaneous multislice, or SMS) and in-plane acceleration (IPA, also called phase acceleration) in diffusion imaging in forty older adults differing in cognitive status. We evaluated a total of 400 scans from five acquisitions: no acceleration (S1P1); SMS=3 with no in-plane acceleration (S3P1); SMS=3 with IPA=2 (S3P2); S6P1; and S6P2. The number of diffusion directions and b-values was kept constant such that acquisition times varied from 21:28 to 3:56. We found that diffusion metrics were highly sensitive to acceleration factor, with a trend towards higher fractional anisotropy (FA) and lower orientation dispersion (OD) with acceleration. The differences between accelerated and unaccelerated acquisitions could be partly explained by the noise amplification (g-factor) and reduced motion. Intraclass correlations (ICCs) of FA and OD in white matter were excellent in both S1P1 and S3P1 (median >0.8), good but lower in S3P2 and S6P1 (medians around 0.70), and poor to fair in S6P2 (medians 0.46 and 0.57). In-plane acceleration decreased ICC, including areas of high susceptibility distortion. In a comparison of mild cognitive impairment versus healthy controls, acceleration tended to reduce group differences, particularly in the fornix, with greater costs in OD than FA. Our results provide guidance regarding the costs of acceleration (possible biases and reduced effect sizes) while also characterizing the benefits (reduced motion, good reliability at higher multiband with no in-plane).

bioengineering↗

bioRxiv2023

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts Through Specific RelA/SpoT Homologs

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RSH enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. A. thaliana mutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting involvement of these synthases in PAMP-triggered innate immunity responses to pathogens within the chloroplast.

biochemistry↗

bioRxiv2023

Vampyroteuthis southchinaseais sp. nov.,a second Recent widely distributed species of Vampyromorpha (Cephalopoda, Coleoidea)

The vampire squid, Vampyroteuthis infernalis Chun, 1903, is currently the only extant species in the family Vampyroteuthidae Chun, 1903. However, specimens from the Gulf of Guinea, Africa, and California suggested the possibility of additional taxa. Here, we report the second species of Vampyroteuthis, collected from the South China Sea, China, which differs from V. infernalis by the tail shape, the lower beak, and the phylogenetic analysis of mitochondrial COI and nuclear large subunit ribosomal DNA (LSU) sequences: V. infernalis present by the lacking of the tail, photophores located approximately one-third of the points between the fins and end, and the lower beak with a broad, short wing; V. pseudoinfernalis Qiu, Liu & Huang, sp. nov. present by an acuate tail, a pair of photophores located at the midpoints between the fins and tail, and a lower beak with a broad, elongate wing.

zoology↗

bioRxiv2023

Inositol pyrophosphate dynamics in yeast reveals control of the PHO starvation program through 1,5-IP8 and the SPX domain of the CDK inhibitor Pho81

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates (IPPs) and SPX receptor domains. Since cells synthesize various IPPs and SPX domains bind them promiscuously, it is unclear whether a specific IPP regulates SPX domains in vivo, or whether multiple IPPs act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the IPP 1-IP7, we now show that the levels of all detectable IPPs of yeast, 1-IP7, 5-IP7 and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses. Significance statementCytosolic Pi is of prime importance for cellular bioenergetics because Pi influences free energy of nucleotide hydrolysis and the metabolite fluxes through glycolysis and oxidative phosphorylation. Eukaryotic cells use the INPHORS pathway to signal Pi via SPX domains and their ligands, inositol pyrophosphates (IP7, IP8), which control Pi homeostasis through a network of target proteins that import, export, store or detoxify Pi. Studies with different systems failed to yield a coherent model on this regulation. We performed the first time-resolved profiling of the full isomer spectrum of inositol pyrophosphates in yeast and dissected the isomer that is relevant to intracellular Pi signaling. Our results can be combined with existing observations from plants, mammals, and other fungi to support a unified model of Pi signaling across all eukaryotic kingdoms, which is in accord with the fundamental importance of Pi management for metabolism.

cell biology↗

bioRxiv2022

β-Lapachone Regulates Mammalian Inositol Pyrophosphate Levels in an NQO1- and Oxygen-dependent Manner

1Inositol pyrophosphates (PP-InsPs) are energetic signalling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involves PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signalling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly {beta}-lapachone ({beta}-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that {beta}-lap treatment caused PP-InsP changes that are oxygen dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that {beta}-lap requires NQO1-bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with {beta}-lap. The data presented here unveil new aspects of {beta}-lap pharmacology and its impact on PP-InsP levels. Our identification of different quinone drugs as modulators of PP-InsP synthesis will allow to better appreciate their overall impact on cellular function. 2 Significance StatementInositol pyrophosphates (PP-InsPs) are messenger molecules regulating various functions in mammals. They are associated with the oxidative stress response, but the underlying mechanism is unclear. We investigate PP-InsP signalling in mammalian cells subjected to reactive oxygen species (ROS). Applying the quinone {beta}-lapachone ({beta}-lap) generated intracellular ROS resulting in decreased PP-InsP levels. This indicates a key role of PP-InsPs in cellular signalling under oxidative stress. Moreover, {beta}-lap mediated PP-InsP signalling required oxygen and the enzyme NAD(P)H:quinone oxidoreductase-1 (NQO1). Since quinone drugs are cytotoxic, our data provide a basis for further investigations into the role of PP-InsPs during quinone-dependent chemotherapies. This is of special relevance since a phase II clinical trial demonstrated {beta}-lap efficacy in a combination chemotherapy against pancreatic cancer.

biochemistry↗

bioRxiv2022

Metabolic consequences of polyphosphate synthesis and imminent phosphate limitation

Cells stabilize intracellular inorganic phosphate (Pi) to compromise between large biosynthetic needs and detrimental bioenergetic effects of Pi. Pi homeostasis in eukaryotes employs SPXs domains, which are receptors for inositol pyrophosphates. We explored how polymerization and storage of Pi in acidocalcisome-like vacuoles supports S. cerevisiae metabolism and how these cells recognize Pi scarcity. Whereas Pi starvation affects numerous metabolic pathways, beginning Pi scarcity affects few metabolites. These include inositol pyrophosphates and ATP, a low-affinity substrate for inositol pyrophosphate-synthesizing kinases. Declining ATP and inositol pyrophosphates may thus be indicators of impending Pi limitation. Actual Pi starvation triggers accumulation of the purine synthesis intermediate 5- aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates Pi-dependent transcription factors. Cells lacking polyphosphate show Pi starvation features already under Pi-replete conditions, suggesting that vacuolar polyphosphate supplies Pi for metabolism even when Pi is abundant. However, polyphosphate deficiency also generates unique metabolic changes that are not observed in starving wildtype cells. Polyphosphate in acidocalcisome-like vacuoles may hence be more than a global phosphate reserve and channel Pi to preferred cellular processes. Abstract importanceCells must strike a delicate balance between the high demand of inorganic phosphate (Pi) for synthesizing nucleic acids and phospholipids, and its detrimental bioenergetic effects by reducing the free energy of nucleotide hydrolysis. The latter may stall metabolism. Therefore, microorganisms manage the import and export of phosphate, its conversion into osmotically inactive inorganic polyphosphates, and their storage in dedicated organelles, acidocalcisomes. Here, we provide novel insights into metabolic changes that cells may use to signal declining phosphate availability in the cytosol and differentiate it from actual phosphate starvation. We also analyze the role of acidocalcisome-like organelles in phosphate homeostasis. This uncovers an unexpected role of the polyphosphate pool in these organelles under phosphate-rich conditions, indicating that its metabolic roles go beyond that of a phosphate reserve for surviving starvation.

cell biology↗

bioRxiv2022

Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly-created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references, and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in Nature. Analysis of feces and the commercial mouse diet suggest the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.

biochemistry↗

bioRxiv2022

Stable isotopomers of myo-inositol to uncover the complex MINPP1-dependent inositol phosphate network

The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers central to a myriad of cellular processes. However, we have an incomplete understanding of InsP metabolism because the available analytical toolset for inositol phosphates is rather limited. Here, we have synthesized and utilized fully and unsymmetrically 13C-labeled myo-inositol and inositol phosphates. These probes were applied in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to further annotate central aspects of InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR - down to individual enantiomers - which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme, that contributes to cellular homeostasis. Full characterization of MINPP1 activity in vitro and in cells, provided a clear picture of this multifunctional phosphatase. Metabolic labeling with stable isotopomers thus constitutes a powerful tool for investigating InsP networks in a variety of different biological contexts.

biochemistry↗

bioRxiv2022

Arabidopsis PFA-DSP-type phosphohydrolases target specific inositol pyrophosphate messengers

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologs display phosphohydrolase activity with a high specificity for the 5-{beta}-phosphate of inositol pyrophosphates. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin-sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-{beta}-phosphate specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

plant biology↗

bioRxiv2022

Generation and characterisation of P. falciparum parasites with a G358S mutation in the PfATP4 Na+ pump and clinically relevant levels of resistance to some PfATP4 inhibitors

Small-molecule inhibitors of PfATP4, a Plasmodium falciparum protein that is believed to pump Na+ out of the parasite while importing H+, are on track to become much-needed new antimalarial drugs. The spiroindolone cipargamin is poised to become the first PfATP4 inhibitor to reach the field, having performed strongly in Phase 1 and 2 clinical trials. Previous attempts to generate cipargamin-resistant parasites in the laboratory have yielded parasites with reduced susceptibility to the drug; however, the highest 50% inhibitory concentration reported to date is 24 nM. Here, we show that P. falciparum parasites can acquire a clinically-significant level of resistance to cipargamin that enables them to withstand micromolar concentrations of the drug. Independent experiments to generate high-level cipargamin resistance using different protocols and strains led to the same change each time - a G358S mutation in PfATP4. Parasites with this mutation showed high-level resistance not only to cipargamin, but also to the dihydroisoquinolone (+)-SJ733. However, for certain other (less clinically advanced) PfATP4-associated compounds the G358S mutation in PfATP4 conferred only moderate resistance or no resistance. The G358S mutation in PfATP4 did not affect parasite susceptibility to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in the Toxoplasma gondii ATP4 homologue (G419S), decreased the sensitivity of the Na+-ATPase activity of ATP4 to inhibition by cipargamin and (+)-SJ733, and decreased the sensitivity of parasites expressing these ATP4 mutations to disruption of parasite Na+ regulation by cipargamin- and (+)-SJ733. The G358S mutation in PfATP4 reduced the affinity of the protein for Na+ and was associated with an increase in the parasites resting cytosolic Na+ concentration; however, no significant defect in parasite growth rate was observed. Our findings suggest that codon 358 in pfatp4 should be monitored closely in the field as a molecular marker for cipargamin resistance, and that PfATP4 inhibitors in clinical development should be tested for their activity against PfATP4G358S parasites.

microbiology↗

bioRxiv2021

Photoaffinity capture compounds to profile the Magic Spot Nucleotide interactomes

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival when confronted with adverse external challenges. In times of antibiotic crisis, a detailed understanding of the stringent response is of critical importance, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium cell lysates applying a family of trifunctional photoaffinity capture compounds. We introduce different MSN probes covering diverse phosphorylation patterns, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of diverse putative MSN receptors both from cytosolic and membrane fractions that, upon validation, unveil new MSN targets. We find, for example, that the dinucleoside polyphosphate hydrolase activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The photoaffinity capture compounds described herein will be useful to identify MSN interactomes under varying conditions and across bacterial species. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/472736v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@15cfc02org.highwire.dtl.DTLVardef@1a4ec1borg.highwire.dtl.DTLVardef@88fd27org.highwire.dtl.DTLVardef@1a42248_HPS_FORMAT_FIGEXP M_FIG Molecular fishing: a family of trifunctional photoaffinity capture compounds enables the identification of Magic Spot Nucleotide receptors by a chemoproteomics approach. C_FIG

biochemistry↗

bioRxiv2020

Analysis of Inositol Phosphate Metabolism by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry (CE-ESI-MS)

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is highly desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables for the first time the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we uncover that there must be unknown inositol synthesis pathways in mammals, highlighting the unique potential of this method to dissect inositol phosphate metabolism and signalling.

biochemistry↗

bioRxiv2019

A Pandas complex adapted for piRNA-guided transposon silencing

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila ovaries, Panoramix (Panx) enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panx functions together with a germline specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), as a ternary complex to suppress transposon expression. Structural and functional analyses demonstrate that dNxf2 binds Panx via its UBA domain, which plays an important role in transposon silencing. Unexpectedly, dNxf2 interacts directly with dNxf1 (TAP), a general nuclear export factor. As a result, dNxf2 prevents dNxf1 from binding to the FG repeats of the nuclear pore complex, a process required for proper RNA export. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. Therefore, we propose that dNxf2 may function as a Pandas (Panoramix-dNxf2 dependent TAP/p15 silencing) complex, which counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates epigenetic gene silencing and heterochromatin formation.

molecular biology↗