Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.     

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA₂β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA₂β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA₂β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA₂β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA₂β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA₂β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.     

A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation

Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1–8–9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1–8–9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1–8–9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.     

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine,a lysine-linked antibody drug conjugate

Antibody–drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla®). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin®), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1.     

Orientation behavior of predaceous ground beetle species in response to volatile emissions identified from yellow starthistle damaged by an invasive slug

We investigated indirect defense in the yellow starthistle (Centaurea solstitialis)–grey garden slug (Deroceras reticulatum)–ground beetle (Pterostichus melanarius and Scaphinotus interruptus) system. In this host plant/herbivore/predator system, the ground beetles are the primary predator of D. reticulatum, the dominant herbivore of the highly invasive weed, C. solstitialis. The aim of our study was to examine the behavioral responses of two species of ground beetle to olfactory stimuli emitted from yellow starthistle damaged by D. reticulatum. The beetle P. melanarius showed a significant preference for the odor of damaged yellow starthistle relative to the odor of intact plants, while S. interruptus did not. Volatiles from D. reticulatum-damaged yellow starthistle were collected and identified as trans-β-farnesene, germacrene D, bicyclogermacrene, and 1,5,9-trimethyl-1,5,9-cyclododecatriene. No quantitative relationship was observed between beetle plant choice or decision time and the level of herbivory. Similarly, there was no relationship between volatile compound relative abundance and level of herbivory, suggesting that our range of leaf damage produces either undetectable semiochemicals or no variation in volatile emission.     

Sequence of a Complete Chicken BG Haplotype Shows Dynamic Expansion and Contraction of Two Gene Lineages with Particular Expression Patterns

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5′ untranslated regions (5′UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.