Iron homeostasis regulates the activity of the microRNA pathway through poly(C)-binding protein 2

MicroRNAs (miRNAs) control gene expression by promoting degradation or repressing translation of target mRNAs. The components of the miRNA pathway are subject to diverse modifications that can modulate the abundance and function of miRNAs. Iron is essential for fundamental metabolic processes, and its homeostasis is tightly regulated. Here we identified iron chelators as a class of activator of the miRNA pathway that could promote the processing of miRNA precursors. We show that cytosolic iron could regulate the activity of the miRNA pathway through poly(C)-binding protein 2 (PCBP2). PCBP2 is associated with Dicer and promotes the processing of miRNA precursors. Cytosolic iron could modulate the association between PCBP2 and Dicer, as well as the multimerization of PCBP2 and its ability to bind to miRNA precursors, which can alter the processing of miRNA precursors. Our findings reveal a role of iron homeostasis in the regulation of miRNA biogenesis.     

Tight complex formation between Cosmc chaperone and its specific client non-native T-synthase leads to enzyme activity and client-driven dissociation

The interaction of the endoplasmic reticulum molecular chaperone Cosmc with its specific client T-synthase (Core 1 β1-3-galactosyltransferase) is required for folding of the enzyme and eventual movement of the T-synthase to the Golgi, but the mechanism of interaction is unclear. Here we show that the lumenal domain of recombinant Cosmc directly interacts specifically in either free form or covalently bound to solid supports with denatured T-synthase but not with the active dimeric form of the enzyme. This leads to formation of a relatively stable complex of Cosmc and denatured T-synthase accompanied by formation of reactivated enzyme in an ATP-independent fashion that is not regulated by redox, calcium, pH, or intermolecular disulfide bond formation. The partly refolded and active T-synthase remains tightly bound noncovalently to Cosmc. Dissociation of T-synthase from the complex is promoted by further interactions of the complex with free forms of either native or non-native T-synthase. Taken together, these results demonstrate a novel mechanism in which Cosmc cycles to bind non-native T-synthase, leading to enzyme activity and release in a client-driven process.     

Recognition of single-stranded nucleic acids by small-molecule splicing modulators

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5′-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.     

Regulation of protein O-glycosylation by the endoplasmic reticulum-localized molecular chaperone Cosmc

Regulatory pathways for protein glycosylation are poorly understood, but expression of branchpoint enzymes is critical. A key branchpoint enzyme is the T-synthase, which directs synthesis of the common core 1 O-glycan structure (T-antigen), the precursor structure for most mucin-type O-glycans in a wide variety of glycoproteins. Formation of active T-synthase, which resides in the Golgi apparatus, requires a unique molecular chaperone, Cosmc, encoded on Xq24. Cosmc is the only molecular chaperone known to be lost through somatic acquired mutations in cells. We show that Cosmc is an endoplasmic reticulum (ER)-localized adenosine triphosphate binding chaperone that binds directly to human T-synthase. Cosmc prevents the aggregation and ubiquitin-mediated degradation of the T-synthase. These results demonstrate that Cosmc is a molecular chaperone in the ER required for this branchpoint glycosyltransferase function and show that expression of the disease-related Tn antigen can result from deregulation or loss of Cosmc function.     

Nucleation of alpha 1-antichymotrypsin polymerization

Alpha(1)-antichymotrypsin is an acute phase plasma protein and a member of the serpin superfamily. We show here that wildtype alpha(1)-antichymotrypsin forms polymers between the reactive center loop of one molecule and the beta-sheet A of a second at a rate that is dependent on protein concentration and the temperature of the reaction. The rate of polymerization was accelerated by seeding with polymers of alpha(1)-antichymotrypsin and a complex of alpha(1)-antichymotrypsin with an exogenous reactive loop peptide but not with reactive loop cleaved alpha(1)-antichymotrypsin or with polymers of other members of the serpin superfamily. Sonication of alpha(1)-antichymotrypsin polymers markedly increased the efficacy of seeding such that polymers were able to form under physiological conditions. Taken together, these data provide the first demonstration that serpin polymerization can result from seeding. This mechanism is analogous to the fibrillization of the Abeta(1-42) peptide and may be important in the deposition of alpha(1)-antichymotrypsin in the plaques of Alzheimer's disease.     

Regulation of myosin-VI targeting to endocytic compartments

Myosin-VI has been implicated in endocytic trafficking at both the clathrin-coated and uncoated vesicle stages. The identification of alternative splice forms led to the suggestion that splicing defines the vesicle type to which myosin-VI is recruited. In contrast to this hypothesis, we find that in all cell types examined, myosin-VI is associated with uncoated endocytic vesicles, regardless of splice form. GIPC, a PDZ-domain containing adapter protein, co-assembles with myosin-VI on these vesicles. Myosin-VI is only recruited to clathrin-coated vesicles in cells that express high levels of Dab2, a clathrin-binding adapter protein. Overexpression of Dab2 is sufficient to reroute myosin-VI to clathrin-coated pits in cells where myosin-VI is normally associated with uncoated vesicles. In normal rat kidney (NRK) cells, which express high endogenous levels of Dab2, splicing of the globular tail domain further modulates targeting of ectopically expressed myosin-VI. Although myosin-VI can be recruited to clathrin-coated pits, we find no requirement for myosin-VI motor activity in endocytosis in NRK cells. Instead, our data suggest that myosin-VI recruitment to clathrin-coated pits may be an early step in the recruitment of GIPC to the vesicle surface.     

Biosynthesis of new indigoid inhibitors of protein kinases using recombinant cytochrome P450 2A6

Glycogen synthase kinase-3 (GSK-3) is a potential drug target for a number of human diseases. Some indigoids have been found to be potent inhibitors of GSK-3, and individual compounds with better activity, specificity, and solubility are desired. In this work, a new disubstituted indigoid generation system was developed with a tryptophanase-deficient Escherichia coli strain as a host to express the human cytochrome P450 2A6 mutant L240C/N297Q, which catalyzes the oxidation of indole to isatin and indoxyl, which in turn react to generate indigoids. Forty-five substituted 1H-indoles from commercial sources were used as substrates in the system, and indigoid mixtures were tested as potential inhibitors of GSK-3. After preliminary screening, cell extracts with high inhibitory activity towards GSK-3alpha/beta were fractionated, and the IC50 values of twelve individual indigoids were measured for GSK-3alpha/beta as well as the protein kinases CDK1/cyclinB and CDK5/p25. Several indigoids, including an indigo, showed stronger inhibition than found in previous work. The most potent towards GSK-3alpha/beta, dimethyl indirubin 5,5'-dicarboxylate (IC50 of 51 nM), was modified by chemical reactions. One product, indirubin 5,5'-dicarboxylic acid 5-methyl ester, inhibited GSK-3alpha/beta with an IC50 of 14 nM and selectivity nearly 40-fold over CDK1 and CDK5. Indirubin-5-5'-dicarbonitrile was also modified to the corresponding 3'-oxime, which had low specificity but showed very high inhibition of all three kinases with IC50 values of 5, 13, and 10 nM towards GSK-3alpha/beta, CDK1, and CDK5, respectively. Thus, this system has the potential to generate new indigoids with therapeutic potential.     

Overexpression of mitogen-activated protein kinase kinase 6 in the heart improves functional recovery from ischemia in vitro and protects against myocardial infarction in vivo

The mitogen-activated protein kinases (MAPK) have been the subject of many studies to identify signaling pathways that promote cell survival or death. In cultured cardiac myocytes, p38 MAPK promotes cell survival or death depending on whether it is activated by mitogen-activated protein kinase kinase 6 (MKK6) or MKK3, respectively. The objectives of the current study were to examine the effects of MKK6-mediated p38 activation in the heart in vivo. Accordingly, we generated transgenic (TG) mice that overexpress wild type MKK6 in a cardiac-restricted manner. Although p38 was about 17-fold more active in TG than non-transgenic (NTG) mouse hearts, TG mouse hearts were morphologically and functionally similar to those of NTG littermates. However, upon transient ischemia followed by reperfusion, the MKK6 TG mouse hearts exhibited significantly better functional recovery and less injury than NTG mouse hearts. Because MKK6 increases levels of the protective small heat shock protein, alpha B-crystallin (alpha BC), in cultured cardiac myocytes, we examined alpha BC levels in the mouse hearts. The level of alpha BC was 2-fold higher in MKK6 TG than NTG mouse hearts. Moreover, ischemia followed by reperfusion induced a 6.4-fold increase in alpha BC levels in the mitochondrial fractions of TG mouse hearts but no increase in alpha BC levels in any of the other fractions analyzed. These alterations in alpha BC expression and localization suggest possible mechanisms of cardioprotection in MKK6 TG mouse hearts.     

Factors influencing synthesis and activity of β-galactosidase inLactobacillus acidophilus

Summary In the type-strainLactobacillus acidophilus ATCC 4356 -galactosidase (-gal) was inducible; lactose, galactose, melibiose and probably maltose, but not glucose, fructose, mannose, sucrose and cellobiose, induced -gal synthesis. Glucose partially inhibited -gal-induction by lactose but not by isopropyl--D-thiogalactoside. -gal synthesis during cell growth was maximal at 0.4% lactose, stimulated by Ca²⁺ but inhibited by Mg²⁺ and Mn²⁺. -gal in the cell-free extract had optimum activity at pH 6.5 and at 45°C. The enzyme activity was stimulated by Mg²⁺, inhibited by Ca²⁺, destroyed by oxidizing agents and protected by reducing agents.     

Bacteriocin plasmids ofPediococcus acidilactici

Summary Pediococcus acidilactici strains E, F and H isolated from fermented sausages produced bacteriocins which were protein in nature and inhibitory to a variety of spoilage and pathogenic microorganisms often encountered in foods. These strains harbored two to three plasmids ranging in size from 7.4 to 40.2 megadaltons. Curing experiments and plasmid profile analysis indicated the involvement of plasmid DNA with bacteriocin activity in all three strains. Carbohydrate fermentation and antibiotic resistance phenotypes did not appear to be associated with bacteriocin plasmids. Both bacteriocin activity and resistance determinants were linked in strain H and mediated by a 7.4-megadalton plasmid, whereas in strains E and F these two traits were not linked.