Targeted inhibition of glucuronidation markedly improves drug efficacy in mice - a model

Finding UDP-glucuronosyltransferases (UGT) require protein kinase C-mediated phosphorylation is important information that allows manipulation of this critical system. UGTs glucuronidate numerous aromatic-like chemicals derived from metabolites, diet, environment and, inadvertently, therapeutics to reduce toxicities. As UGTs are inactivated by downregulating PKCs with reversibly-acting dietary curcumin, we determined the impact of gastro-intestinal glucuronidation on free-drug uptake and efficacy using immunosuppressant, mycophenolic acid (MPA), in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversibly and reversibly dephosphorylation by PKC-specific inhibitor calphostin-C and general-kinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral curcumin-administration to mice reversibly inhibited glucuronidation in GI-tissues. Finally, successive oral administration of curcumin and MPA to antigen-treated mice increased serum free MPA and immunosuppression up to 9-fold. Results indicate targeted inhibition of GI glucuronidation in mice markedly improved free-chemical uptake and efficacy using MPA as a model.     

Intracellular mechanics of migrating fibroblasts

Cell migration is a highly coordinated process that occurs through the translation of biochemical signals into specific biomechanical events. The biochemical and structural properties of the proteins involved in cell motility, as well as their subcellular localization, have been studied extensively. However, how these proteins work in concert to generate the mechanical properties required to produce global motility is not well understood. Using intracellular microrheology and a fibroblast scratch-wound assay, we show that cytoskeleton reorganization produced by motility results in mechanical stiffening of both the leading lamella and the perinuclear region of motile cells. This effect is significantly more pronounced in the leading edge, suggesting that the mechanical properties of migrating fibroblasts are spatially coordinated. Disruption of the microtubule network by nocodazole treatment results in the arrest of cell migration and a loss of subcellular mechanical polarization; however, the overall mechanical properties of the cell remain mostly unchanged. Furthermore, we find that activation of Rac and Cdc42 in quiescent fibroblasts elicits mechanical behavior similar to that of migrating cells. We conclude that a polarized mechanics of the cytoskeleton is essential for directed cell migration and is coordinated through microtubules.     

Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network

Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches.     

Cysteine 981 of the human insulin receptor is required for covalent cross-linking between beta-subunit and a thiol-reactive membrane-associated protein

The cytoplasmic domain of the insulin receptor (IR) beta-subunit contains cysteine (Cys) residues whose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctional cross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins that possess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type human IR, C-terminally truncated receptors, or mutant receptors with Cys --> Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complex between the wild-type human receptor beta-subunit and molecule X, a thiol-reactive membrane-associated protein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulin had only a modest effect in this process. Western blot analysis revealed that the receptor alpha-subunit was not present in the beta-X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylation of the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, that lacks the equivalent of human Cys(981), failed to react with BMH. Finally, no covalent association between IR beta-subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treated cells expressing the wild-type human IR. These results demonstrate a striking difference in reactivity among the cytoplasmic IR beta-subunit thiols and clearly show that Cys(981) of human IR beta-subunit is in close proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulated conditions.     

Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.     

Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5'' but not the 3'' splice site inhibit intron processing in Nicotiana plumbaginifolia.

We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.     

Characterization and partial purification of an insulinase from Neurospora crassa.

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.     

Inactivation of splicing factors in HeLa cells subjected to heat shock

The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.     

Modulation of RNA splicing as a potential treatment for cancer

Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant. This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs.?SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ?7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.