MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.     

Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.     

The protective effects of MSC‐EXO against pulmonary hypertension through regulating Wnt5a/BMP signalling pathway

The aim of the study was to explore the mechanism of mesenchymal stem cell‐derived exosomes (MSC‐EXO) to protect against experimentally induced pulmonary hypertension (PH). Monocrotaline (MCT)‐induced rat model of PH was successfully established by a single intraperitoneal injection of 50 mg/kg MCT, 3 weeks later the animals were treated with MSC‐EXO via tail vein injection. Post‐operation, our results showed that MSC‐EXO could significantly reduce right ventricular systolic pressure (RVSP) and the right ventricular hypertrophy index, attenuate pulmonary vascular remodelling and lung fibrosis in vivo. In vitro experiment, the hypoxia models of pulmonary artery endothelial cell (PAEC) and pulmonary vascular smooth muscle cell (PASMC) were used. We found that the expression levels of Wnt5a, Wnt11, BMPR2, BMP4 and BMP9 were increased, but β‐catenin, cyclin D1 and TGF‐β1 were decreased in MSC‐EXO group as compared with MCT or hypoxia group in vivo or vitro. However, these increased could be blocked when cells were transfected with Wnt5a siRNA in vitro. Taken together, these results suggested that the mechanism of MSC‐EXO to prevent PH vascular remodelling may be via regulation of Wnt5a/BMP signalling pathway.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Effect of thiostrepton and 3′-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis

The effect of the antibiotics thiostrepton and micrococcin on EF-T_u-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3′-terminus of aminoacyl-tRNA)(System I) or by methanol (‘uncoupled GTPase’, System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-T_u·GTP·70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the K^GTP_m is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases V^GTP_max, whereas K^GTP_m remains unaffected. Micrococcin is without any effect in both systems. The ‘uncoupled GTPase’ (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-T_u site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-T_u for GTP.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

Heliothrix oregonensis,gen. nov., sp. nov., a phototrophic filamentous gliding bacterium containing bacteriochlorophyll a

An unusual filamentous, gliding bacterium was found in a few hot springs in Oregon where it formed a nearly unispecific top layer of microbial mats. It contained a bacteriochlorophyll a-like pigment and an abundance of carotenoids. There were no chlorosomes or additional chlorophylls. The organism was aerotolerant and appeared to be photoheterotrophic. It was successfully co-cultured with an aerobic chemoheterotroph in a medium containing glucose and casamino acids. Although it has many characteristics in common with the genus Chloroflexus, the lack of chlorosomes and bacteriochlorophyll c and the aerobic nature of this organism indicate that it should be placed in a new genus. This conclusion is supported by 5S rRNA nucleotide sequence data.     

Bone marrow-derived mesenchymal stem cells repair severe acute pancreatitis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling

BackgroundSevere acute pancreatitis (SAP) is associated with high morbidity and mortality. Bone marrow mesenchymal stem cells (BMSCs) have shown obvious protective effect on SAP. However, little is known about the underlying mechanism. The objective of this study is to unravel the role and regulatory mechanism of miR-181a-5p in BMSCs-mediated pancreatic repair.MethodsBMSCs were isolated from Sprague-Dawley rats and characterized by flow cytometry and Oil Red O staining. Sodium taurocholate- and caerulein-induced models were used as SAP models in vivo and in vitro, respectively. Pancreatic injury were evaluated by H&E and histopathological analysis, as well as by measuring levels of amylase, lipase and cytokines. qRT-PCR and western blotting were performed to detect the level of miR-181a-5p and the protein levels of PTEN/Akt, respectively. ELISA was conducted to detect the levels of TNF-α, IL-1β, IL-6, angiopoietin, IL-4, IL-10 and TGF-β1. The apoptotic rate of AR42 J cells was quantitated by concurrent staining with Annexin-V-FITC and PI.ResultsBMSCs significantly attenuated pancreatic injury in SAP rats by reducing inflammatory infiltration and necrosis, and this effect was abolished by CXCR4 agonist AMD3100. ADM3100 exhibited more severe pancreatic injury and decreased miR-181a-5p levels in the pancreas and serum compared to SAP group. Overexpression of miR-181a-5p in BMSCs (BMSCs-miR-181a-5p) markedly potentiated the protective effect of BMSCs by reducing histological damage and levels of amylase and lipase. Moreover, BMSCs-miR-181a-5p dramatically reduced levels of angiopoietin, TNF-α, IL-1β and IL-6, but induced the levels of IL-4 and IL-10. In caerulein-treated AR42 J cells, co-culturing of BMSCs-miR-181a-5p alleviated caerulein-induced increase of amylase and lipase, and apoptosis via PTEN/Akt/TGF-β1 signaling.ConclusionBMSCs alleviate SAP and reduce inflammatory responses and apoptosis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling. Hence, BMSCs-miR-181a-5p could serve as potential therapeutic target for SAP.