Messenger RNA delivery to mitoribosomes - hints from a bacterial toxin.

In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome. When the small subunit fails to assemble, however, the stability of mt-mRNA is only marginally affected, and under these conditions, the LRPPRC/SLIRP RNA-binding complex has been implicated in maintaining mt-mRNA stability. Here, we exploit the activity of a bacterial ribotoxin, VapC20, to show that in the absence of the large mitoribosomal subunit, mt-mRNA species are selectively lost. Further, if the small subunit is also depleted, the mt-mRNA levels are recovered. As a consequence of these data, we suggest a natural pathway for loading processed mt-mRNA onto the mitoribosome.

The Fungal Iron Chelator Desferricoprogen Inhibits Atherosclerotic Plaque Formation.

Hemoglobin, heme and iron are implicated in the progression of atherosclerosis. Therefore, we investigated whether the hydrophobic fungal iron chelator siderophore, desferricoprogen (DFC) inhibits atherosclerosis. DFC reduced atherosclerotic plaque formation in ApoE-/- mice on an atherogenic diet. It lowered the plasma level of oxidized LDL (oxLDL) and inhibited lipid peroxidation in aortic roots. The elevated collagen/elastin content and enhanced expression of adhesion molecule VCAM-1 were decreased. DFC diminished oxidation of Low-density Lipoprotein (LDL) and plaque lipids catalyzed by heme or hemoglobin. Formation of foam cells, uptake of oxLDL by macrophages, upregulation of CD36 and increased expression of TNF-α were reduced by DFC in macrophages. TNF-triggered endothelial cell activation (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecules (ICAMs), E-selectin) and increased adhesion of monocytes to endothelium were attenuated. The increased endothelial permeability and intracellular gap formation provoked by TNF-α was also prevented by DFC. DFC acted as a cytoprotectant in endothelial cells and macrophages challenged with a lethal dose of oxLDL and lowered the expression of stress-responsive heme oxygenase-1 as sublethal dose was employed. Saturation of desferrisiderophore with iron led to the loss of the beneficial effects. We demonstrated that DFC accumulated within the atheromas of the aorta in ApoE-/- mice. DFC represents a novel therapeutic approach to control the progression of atherosclerosis.

Poly-Saturated Dolichols from Filamentous Fungi Modulate Activity of Dolichol-Dependent Glycosyltransferase and Physical Properties of Membranes.

 Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.

Snapshots of C-S Cleavage in Egt2 Reveals Substrate Specificity and Reaction Mechanism.

Sulfur incorporation in the biosynthesis of ergothioneine, a histidine thiol derivative, differs from other well-characterized transsulfurations. A combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation and a subsequent pyridoxal 5'-phosphate (PLP)-mediated C-S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine. In this study, we structurally and mechanistically characterized a PLP-dependent C-S lyase Egt2, which mediates the sulfoxide C-S bond cleavage in ergothioneine biosynthesis. A cation-π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C-S lyase reaction cycle, including a labile sulfenic intermediate captured in Egt2 crystals. Chemical trapping and high-resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis.

The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates.

DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich "C-tail" that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (kcat/KM) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or "threshold" number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.

Assembly of eIF3 Mediated by Mutually Dependent Subunit Insertion.

Eukaryotic initiation factor 3 (eIF3), an essential multi-protein complex involved in translation initiation, is composed of 12 tightly associated subunits in humans. While the overall structure of eIF3 is known, the mechanism of its assembly and structural consequences of dysregulation of eIF3 subunit expression seen in many cancers is largely unknown. Here we show that subunits in eIF3 assemble into eIF3 in an interdependent manner. Assembly of eIF3 is governed primarily by formation of a helical bundle, composed of helices extending C-terminally from PCI-MPN domains in eight subunits. We propose that, while the minimal subcomplex of human-like eIF3 functional for translation initiation in cells consists of subunits a, b, c, f, g, i, and m, numerous other eIF3 subcomplexes exist under circumstances of subunit over- or underexpression. Thus, eIF3 subcomplexes formed or "released" due to dysregulated subunit expression may be determining factors contributing to eIF3-related cancers.

Tubulin tail sequences and post-translational modifications regulate closure of mitochondrial voltage-dependent anion channel (VDAC).

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and ß-tubulin subunits. It was unclear whether the α- and ß-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the ß-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.

Bioinformatic and biochemical characterizations of C-S bond formation and cleavage enzymes in the fungus Neurospora crassa ergothioneine biosynthetic pathway.

Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C-S bond formation and a PLP-mediated C-S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C-S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible.

Role of Saccharomyces cerevisiae Trk1 in stabilization of intracellular potassium content upon changes in external potassium levels.

Saccharomyces cerevisiae cells are able to grow at very different potassium concentrations adapting its intracellular cation levels to changes in the external milieu. Potassium homeostasis in wild type cells resuspended in media with low potassium is an example of non-perfect adaptation since the same intracellular concentration is not approached irrespective of the extracellular levels of the cation. By using yeasts lacking the Trk1,2 system or expressing different versions of the mutated main plasma membrane potassium transporter (Trk1), we show that Trk1 is not essential for adaptation to potassium changes but the dynamics of potassium loss is very different in the wild type and in trk1,2 mutant or in yeasts expressing Trk1 versions with highly impaired transport characteristics. We also show that the pattern here described can be also fulfilled by heterologous expression of NcHAK1, a potassium transporter not belonging to the TRK family. Hyperpolarization and cationic drugs sensitivity in mutants with defective transport capacity provide additional support to the hypothesis of connections between the activity of the Trk system and the plasma membrane H(+) ATPase (Pma1) in the adaptive process.

The amino acids surrounding the flavin 7a-methyl group determine the UVA spectral features of a LOV protein.

Flavin-binding light, oxygen, and voltage (LOV) domains are UVA/blue-light-sensing protein units that form a reversible flavin mononucleotide-cysteine adduct upon light induction. In their dark-adapted state, LOV domains exhibit the typical spectral features of fully oxidized riboflavin derivatives. A survey on the absorption spectra of various LOV domains revealed that the UVA spectral range is the most variable region (whereas the absorption band at 450 nm is virtually unchanged), showing essentially two distinct patterns found in plant phototropin LOV1 and LOV2 domains, respectively. In this work, we have identified a residue directly interacting with the isoalloxazine methyl group at C(7a) as the major UVA spectral tuner. In YtvA from Bacillus subtilis, this amino acid is threonine 30, and its mutation into apolar residues converts the LOV2-like spectrum of native YtvA into a LOV1-like pattern. Mutation T30A also accelerates the photocycle ca. 4-fold. Together with control mutations at different positions, our results experimentally confirm the previously calculated direction of the transition dipole moment for the UVA ππ* state and identify the mechanisms underlying spectral tuning in the LOV domains.

Nonself recognition through intermolecular disulfide bond formation of ribonucleotide reductase in neurospora.

Type I ribonucleotide reductases (RNRs) are conserved across diverse taxa and are essential for the conversion of RNA into DNA precursors. In Neurospora crassa, the large subunit of RNR (UN-24) is unusual in that it also has a nonself recognition function, whereby coexpression of Oak Ridge (OR) and Panama (PA) alleles of un-24 in the same cell leads to growth inhibition and cell death. We show that coexpressing these incompatible alleles of un-24 in N. crassa results in a high molecular weight UN-24 protein complex. A 63-amino-acid portion of the C terminus was sufficient for un-24(PA) incompatibility activity. Redox active cysteines that are conserved in type I RNRs and essential for their catalytic function were found to be required for incompatibility activity of both UN-24(OR) and UN-24(PA). Our results suggest a plausible model of un-24 incompatibility activity in which the formation of a complex between the incompatible RNR proteins is potentiated by intermolecular disulfide bond formation.

Structure of a light-activated LOV protein dimer that regulates transcription.

Light, oxygen, or voltage (LOV) protein domains are present in many signaling proteins in bacteria, archaea, protists, plants, and fungi. The LOV protein VIVID (VVD) of the filamentous fungus Neurospora crassa enables the organism to adapt to constant or increasing amounts of light and facilitates proper entrainment of circadian rhythms. Here, we determined the crystal structure of the fully light-adapted VVD dimer and reveal the mechanism by which light-driven conformational change alters the oligomeric state of the protein. Light-induced formation of a cysteinyl-flavin adduct generated a new hydrogen bond network that released the amino (N) terminus from the protein core and restructured an acceptor pocket for binding of the N terminus on the opposite subunit of the dimer. Substitution of residues critical for the switch between the monomeric and the dimeric states of the protein had profound effects on light adaptation in Neurospora. The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions.

Illuminating solution responses of a LOV domain protein with photocoupled small-angle X-ray scattering.

The PAS-LOV domain is a signal-transducing component found in a large variety of proteins that is responsible for sensing different stimuli such as light, oxygen, and voltage. The LOV protein VVD regulates blue light responses in the filamentous fungi Neurospora crassa. Using photocoupled, time-resolved small-angle X-ray scattering, we extract the solution protein structure in both dark-adapted and light-activated states. Two distinct dark-adapted conformations are detected in the wild-type protein: a compact structure that corresponds to the crystal structure of the dark-state monomer as well as an extended structure that is well modeled by introducing conformational disorder at the N-terminus of the protein. These conformations are accentuated in carefully selected variants, in which a key residue for propagating structural transitions, Cys71, has been mutated or oxidized. Despite different dark-state conformations, all proteins form a common dimer in response to illumination. Taken together, these data support a reaction scheme that describes the mechanism for light-induced dimerization of VVD. Envelope reconstructions of the transient light-state dimer reveal structures that are best described by a parallel arrangement of subunits that have significantly changed conformation compared to the crystal structure.

Properties of the kinesin-3 NcKin3 motor domain and implications for neck function.

Members of the Kinesin-3 family are microtubule motors involved in the transport of membranous cargo. NcKin3 from the fungus Neurospora crassa is dimeric but inactivates one of its motor heads to generate nonprocessive motility. To determine how one of the heads is inactivated, we investigated truncated monomeric constructs. None of the constructs generated processive single-molecule motility, and multimotor velocities depended linearly on the number of residues remaining in the neck. The kinetic analysis suggests futile ATP hydrolysis cycles, because a representative monomer showed a faster ATP turnover than the dimer while supporting slower motility. The K(0.5,MT) was 70-fold lower, the microtubule-bound portion of the kinetic cycle eight-fold longer and the microtubule detachment rate almost 15-fold slower than that of the dimer. Moreover, the monomer's microtubule-dependent ADP release occurred three-fold to four-fold faster than k(cat) (125 versus 34 s(-1)), whereas phosphate release was approximately equally fast (29 s(-1)). A dimeric construct containing a structure-breaking insert between motor head and neck showed a similar behaviour. These data suggest that the heads of the wild-type NcKin3 motor are strictly coupled via the neck domain, and that the dimeric structure is required for proper detachment after one ATPase cycle. This is the first direct comparison of a monomeric Kinesin-3 with its dimeric full-length counterpart, and the kinetic changes observed here may also apply to other Kinesin-3 motors.

Microtubule dynamics and the role of molecular motors in Neurospora crassa.

Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leading hyphae and germ tubes of Neurospora crassa expressing beta-tubulin-GFP. Microtubule polymerization rates in hyphae of N. crassa were much faster than those previously reported in any other eukaryotic organism. In order to address the roles of motor proteins in microtubule dynamic instability in N. crassa, the microtubule-motor mutant strains, Deltankin and ro-1, were examined. Polymerization and depolymerization rates in leading hyphae of these strains were reduced by one half relative to the wild type. Furthermore, microtubules in germ tubes of wild type and microtubule-motor mutants exhibited similar dynamic characteristics as those in hyphae of mutant strains. Small microtubule fragments exhibiting anterograde and retrograde motility were present in leading hyphae of all strains and germ tubes of wild-type strains. Our data suggest that microtubule motors play important roles in regulating microtubule dynamic instability in leading hyphae but not in germ tubes.

Conformational switching in the fungal light sensor Vivid.

The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).

Role of the conserved cysteine residues of the 11.5 kDa subunit in complex I catalytic properties.

Mitochondrial complex I exists as a mixture of two inter-convertible forms: active (A) and de-activated (D), the latter being sensitive to SH-modifying compounds. To investigate if the conserved cysteine-rich 11.5 kDa subunit of Neurospora crassa complex I is involved in this process, we subjected the corresponding genomic DNA to site-directed mutagenesis. The four cysteine residues of the subunit were separately substituted with serine residues and the resulting proteins were independently expressed in a null-mutant strain. All of the obtained mutant strains were able to assemble a complex I with similar kinetic properties to those observed in the wild-type enzyme, indicating that none of the cysteine residues of the 11.5 kDa protein is individually relevant for the A/D transition process. Diminished amounts of assembled complex I seem to be the major effect of these specific mutations. The cysteine residues are likely important to the acquisition and stabilization of the correct 11.5 kDa protein conformation and this is reflected in the assembly/stability of complex I.

Structural analysis of the ZEN-4/CeMKLP1 motor domain and its interaction with microtubules.

The centralspindlin complex is required for the assembly and maintenance of the central spindle during late anaphase and the completion of cytokinesis. It is composed of two copies each of the kinesin-like protein ZEN-4, a Caenorhabditis elegans MKLP-1 (Kinesin-6 family), and the RhoGAP CYK-4. By using cryo-electron microscopy and helical 3D reconstruction, we are investigating the structural features of the interactions between monomeric and dimeric motor domain constructs of ZEN-4 and microtubules. We have calculated helically averaged 3D maps of microtubules decorated with ZEN-4 motor domain in the presence of AMP-PNP, ADP, ADP-AlF(4)(-), and nucleotide-free conditions. We used statistical difference mapping to compare these maps among each other and to related maps obtained from microtubules decorated with a well-characterized Kinesin-1 motor domain from Neurospora crassa. Thereby, we found distinct structural features in microtubule-ZEN-4 complexes that may directly relate to the functional properties of ZEN-4 and centralspindlin. Furthermore, we investigated the location, structure, and function of a highly conserved extension of approximately 50 residues unique to the Kinesin-6 subfamily, located in the motor core loop6/beta4 region.

A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core.

The neck domain of fungal conventional kinesins displays characteristic properties which are reflected in a specific sequence pattern. The exchange of the strictly conserved Tyr 362, not present in animals, into Lys, Cys or Phe leads to a failure to dimerize. The destabilizing effect is confirmed by a lower coiled-coil propensity of mutant peptides. Whereas the Phe substitution has only a structural effect, the Lys and Cys replacements lead to dramatic kinetic changes. The steady state ATPase is 4- to 7-fold accelerated, which may be due to a faster microtubule-stimulated ADP release rate. These data suggest that an inhibitory effect of the fungal neck domain on the motor core is mediated by direct interaction of the aromatic ring of Tyr 362 with the head, whereas the OH group is essential for dimerization. This is the first demonstration of a direct influence of the kinesin neck region in regulation of the catalytic activity.

Characterization of DNA binding and the cysteine rich region of SRE, a GATA factor in Neurospora crassa involved in siderophore synthesis.

Several homologous genes encoding proteins involved in regulating siderophore synthesis in fungi have been isolated, including the sre gene from the filamentous fungus Neurospora crassa. We present data that further characterize SRE and provide new insights into the regulation of iron homeostasis in Neurospora. SRE is a member of the GATA factor family, which is comprised of transcription factors that contain either one or two zinc finger motifs that recognize and bind to GATA-containing DNA sequences. Results from electrophoretic mobility shift assays demonstrate that SRE binds with high affinity to a DNA probe containing the iron response element from the sid1 promoter from Ustilago. SRE binding to DNA was demonstrated to be zinc-dependent. Moreover, changes in the spacing between two GATA sites altered the DNA binding affinity of SRE. Mutants of highly conserved cysteine residues present in SRE and homologous proteins were created by site-directed mutagenesis. The combined results of mobility shift assays, siderophore synthesis assays, and ornithine oxygenase enzyme activity determinations demonstrate that these mutants with cysteine substitutions have a dominant repressor phenotype.