Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris

Hydrophobins are small highly surface-active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression host Pichia pastoris. One-, 2-, and 3-copy-HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression of PDI1 or ERO1 caused insignificant changes in HFBI secretion in 1- and 2-copy strains, but a statistically significant HFBI secretion increase in 3-copy strain. KAR2 overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3-copy-HFBI secreting 22±1.6 fold more than the 1-copy-HFBI/no chaperone strain. The highest increase was seen in 3-copy-HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1-copy-HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small-scale format used in this study.     

Effects of the multi-kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA-approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi-targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild-type (wt) FLT3-expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.     

Segregating the Effects of Seed Traits and Common Ancestry of Hardwood Trees on Eastern Gray Squirrel Foraging Decisions

The evolution of specific seed traits in scatter-hoarded tree species often has been attributed to granivore foraging behavior. However, the degree to which foraging investments and seed traits correlate with phylogenetic relationships among trees remains unexplored. We presented seeds of 23 different hardwood tree species (families Betulaceae, Fagaceae, Juglandaceae) to eastern gray squirrels (Sciurus carolinensis), and measured the time and distance travelled by squirrels that consumed or cached each seed. We estimated 11 physical and chemical seed traits for each species, and the phylogenetic relationships between the 23 hardwood trees. Variance partitioning revealed that considerable variation in foraging investment was attributable to seed traits alone (27–73%), and combined effects of seed traits and phylogeny of hardwood trees (5–55%). A phylogenetic PCA (pPCA) on seed traits and tree phylogeny resulted in 2 “global” axes of traits that were phylogenetically autocorrelated at the family and genus level and a third “local” axis in which traits were not phylogenetically autocorrelated. Collectively, these axes explained 30–76% of the variation in squirrel foraging investments. The first global pPCA axis, which produced large scores for seed species with thin shells, low lipid and high carbohydrate content, was negatively related to time to consume and cache seeds and travel distance to cache. The second global pPCA axis, which produced large scores for seeds with high protein, low tannin and low dormancy levels, was an important predictor of consumption time only. The local pPCA axis primarily reflected kernel mass. Although it explained only 12% of the variation in trait space and was not autocorrelated among phylogenetic clades, the local axis was related to all four squirrel foraging investments. Squirrel foraging behaviors are influenced by a combination of phylogenetically conserved and more evolutionarily labile seed traits that is consistent with a weak or more diffuse coevolutionary relationship between rodents and hardwood trees rather than a direct coevolutionary relationship.     

AIF deficiency compromises oxidative phosphorylation

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.     

Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors

A homogenous TR-FRET-based in vitro coupling assay for the MAP3Ks–MEK1–ERK2 kinase cascade was established and was used to screen for inhibitors of the ERK/MAPK pathway. A series of coumarin derivatives were identified from the screen. These compounds potently inhibit the activation of the unactivated human MEK1 by upstream MAP3Ks (including BRAF and COT), but do not inhibit the activity of the activated MEK1. In addition, the potency of these compounds in inhibiting MEK1 activation is not affected by varying the ATP concentration, suggesting that these inhibitors are not competitive with ATP. As expected, the coumarin compounds potently inhibit LPS-induced TNF production and ERK phosphorylation in THP-1 cells, with the most potent compound having an IC₅₀ of 90 nM. Molecular modeling studies suggest that these coumarins bind to an allosteric site in the inactive conformation of MEK1. This site has been shown to be utilized by the biarylamine series of MEK inhibitors such as PD318088. Very interestingly, the identified coumarin derivatives are almost identical to a series of inhibitors recently reported that block LPS-induced TNF production. Our findings have therefore raised the possibility that other naturally occurring or synthetic coumarins with anti-cancer and anti-inflammatory activities might exert their biological function through the inhibition of MEK1.     

Primary structural features of the<Emphasis Type="Italic"> S</Emphasis> haplotype-specific F-box protein,SFB, in<Emphasis Type="Italic"> Prunus</Emphasis>

The gene SFB encodes an F-box protein that has appropriate S-haplotype-specific variation to be the pollen determinant in the S-RNase-based gametophytic self-incompatibility (GSI) reaction in Prunus (Rosaceae). To further characterize Prunus SFB, we cloned and sequenced four additional alleles from sweet cherry (P. avium), SFB ¹ , SFB ² , SFB ⁴ , and SFB ⁵ . These four alleles showed haplotype-specific sequence diversity similar to the other nine SFB alleles that have been cloned. In an amino acid alignment of Prunus SFBs, including the four newly cloned alleles, 121 out of the 384 sites were conserved and an additional 65 sites had only conservative replacements. Amino acid identity among the SFBs ranged from 66.0% to 82.5%. Based on normed variability indices (NVI), 34 of the non-conserved sites were considered to be highly variable. Most of the variable sites were located at the C-terminal region. A window-averaged plot of NVI indicated that there were two variable and two hypervariable regions. These variable and hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the GSI reaction. Evidence of positive selection was detected using maximum likelihood methods with sites under positive selection concentrated in the variable and hypervariable regions.K. Ikeda and B. Igic contributed equally to this paperNucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers AB111518, AB111519, AB111520, and AB111521, for SFB ¹, SFB ², SFB ⁵, and SFB ⁴, respectively     

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.