In vivo biotinylated calpastatin improves the affinity purification of human m-calpain

Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies. © 2018 The Author

N-glycosylation site analysis reveals sex-related differences in protein N-glycosylation in the rice brown planthopper (Nilaparvata lugens)

Glycosylation is a common modification of proteins and critical for a wide range of biological processes. Differences in protein glycosylation between sexes have already been observed in humans, nematodes and trematodes, and have recently also been reported in the rice pest insect Nilaparvata lugens. Although protein N-glycosylation in insects is nowadays of high interest because of its potential for exploitation in pest control strategies, the functionality of differential N-glycosylation between sexes is yet unknown. In this study, therefore, the occurrence and role of sex-related protein N-glycosylation in insects were examined. A comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes at the glycopeptide level. N-glycopeptide enrichment via lectin capturing using the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans, resulted in the identification of over 1300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosylation sites were shown to be derived from proteins with a putative role in insect reproduction. In addition, differential glycan composition between males and females was observed for proteins shared across sexes. Both lectin blotting experiments as well as transcript expression analyses with complete insects and insect tissues confirmed the observed differences in N-glycosylation of proteins between sexes. In conclusion, this study provides further evidence for protein N-glycosylation to be sex-related in insects. Furthermore, original data on N-glycosylation sites of N. lugens adults are presented, providing novel insights into planthopper's biology and information for future biological pest control strategies

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor a-chain (IL-7Ra) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Ra extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency

Simple peptide quantification approach for MS-based proteomics quality control

Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with the sample preparation protocol, in particular, after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in the MS-loading solvent, consuming only 2 mu L of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 mu g as an optimal peptide amount to be injected into this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control

The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes

The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein.

BACKGROUND: Regions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism. METHODS: Stable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis. RESULTS: HIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated. CONCLUSIONS: For the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown

The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein.

BACKGROUND: Regions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism. METHODS: Stable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis. RESULTS: HIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated. CONCLUSIONS: For the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown

Glutaredoxin GRXS17 associates with the cytosolic iron-sulfur cluster assembly pathway

Cytosolic monothiol glutaredoxins (GRXs) are required in iron-sulfur (Fe-S) cluster delivery and iron sensing in yeast and mammals. In plants, it is unclear whether they have similar functions. Arabidopsis (Arabidopsis thaliana) has a sole class II cytosolic monothiol GRX encoded by GRXS17. Here, we used tandem affinity purification to establish that Arabidopsis GRXS17 associates with most known cytosolic Fe-S assembly (CIA) components. Similar to mutant plants with defective CIA components, grxs17 loss-of-function mutants showed some degree of hypersensitivity to DNA damage and elevated expression of DNA damage marker genes. We also found that several putative Fe-S client proteins directly bind to GRXS17, such as XANTHINE DEHYDROGENASE1 (XDH1), involved in the purine salvage pathway, and CYTOSOLIC THIOURIDYLASE SUBUNIT1 and CYTOSOLIC THIOURIDYLASE SUBUNIT2, both essential for the 2-thiolation step of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNAs. Correspondingly, profiling of the grxs17-1 mutant pointed to a perturbed flux through the purine degradation pathway and revealed that it phenocopied mutants in the elongator subunit ELO3, essential for the mcm5 tRNA modification step, although we did not find XDH1 activity or tRNA thiolation to be markedly reduced in the grxs17-1 mutant. Taken together, our data suggest that plant cytosolic monothiol GRXs associate with the CIA complex, as in other eukaryotes, and contribute to, but are not essential for, the correct functioning of client Fe-S proteins in unchallenged conditions

DYn-2 based identification of Arabidopsis sulfenomes

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana

Decoding the Folding of Burkholderia glumae Lipase: Folding Intermediates En Route to Kinetic Stability

The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier