Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

The cell relies on direct interactions among proteins and compartments called organelles to stay alive. Manipulating these interactions allows researchers to control a wide variety of cell behaviors. A system called 'Magnets' uses light to trigger interactions between proteins. Magnets uses a segment of a protein called Vivid from a common bread mold that responds to light. When light shines on two of these segments, it causes them to bind together, in a process known as dimerization. In the Magnets system, Vivid segments are attached to specific proteins or organelles. By using light, researchers can force their target molecules to come together and trigger signals that can change cell behavior. However, the Magnets system has limitations: its stability and low efficiency mean that the cells need to be kept at low temperatures and that several copies of Vivid are needed. These conditions can interfere with the activity of the target proteins. To expand the technique, Benedetti et al. added mutations to make the Vivid protein more similar to proteins found in fungi that thrive at temperatures around 50°C. These changes meant that the enhanced system could work at body temperature in mammals. Further mutations at the interface between the two Vivid segments improved the efficiency of dimerization. This enhanced version was put to the test in different applications, including delivering proteins to different organelles and bringing organelles together. The enhanced Magnets system should enable researchers to control a greater variety of signaling events in the cell. In addition, the methodology established for improving the efficiency of the Magnets system could be useful to researchers working on other proteins.

Light-regulated synthesis of extra- and intracellular enzymes related to wood degradation by the white rot fungus Cerrena unicolor during solid-state fermentation on ash sawdust-based medium.

The light-dependent metabolism of the white rot basidiomycete Cerrena unicolor FCL139 has already been demonstrated using transcriptomic and Biolog-based approaches. To further analyze the influence of light on C. unicolor wood degradation, we measured the activity of an array of CAZymes (carbohydrate-active enzymes) and enzymes involved in the redox system of fungal cells associated with lignolysis. Extra- and intracellular enzymatic extracts were obtained from solid-state ash sawdust C. unicolor cultures cultivated for 14 days under red, blue, green, or white light conditions, or in the dark. Light greatly influenced the synthesis of MnP, total cellulases, endo-1,4-ß-glucanase, endo-1,4-ß-xylanase, catalase, and superoxide dismutase. The production of MnP and catalase was evidently stimulated by white light. It is also worth noticing that blue light caused a gradual increase in the activity of total cellulases throughout the entire period of C. unicolor growth. Moreover, endo-1,4-ß-glucanase showed the highest activity on day 13 of fungus cultivation and the production of laccase and ß-glucosidase appeared to be the least influenced by light. However, the strongest activity of the endo-1,4-ß-xylanase was observed in the dark. It seemed that light not only influenced the regulation of the synthesis of the wood-degrading enzymes at different levels, but also acted indirectly by affecting production of enzymes managing harmful lignin by-products causing oxidative stress. The ability of the fungus to decompose woody plant material is clearly influenced by environmental factors.

Short-Lived Radical Intermediates in the Photochemistry of Glucose Oxidase.

Glucose oxidase is a flavoprotein that is relatively well-studied as a physico-chemical model system. The flavin cofactor is surrounded by several aromatic acid residues that can act as direct and indirect electron donors to photoexcited flavin. Yet, the identity of the photochemical product states is not well established. We present a detailed full spectral reinvestigation of this issue using femtosecond fluorescence and absorption spectroscopy. Based on a recent characterization of the unstable tyrosine cation radical TyrOH•+ , we now propose that the primary photoproduct involves this species, which was previously not considered. Formation of this product is followed by competing charge recombination and radical pair stabilization reactions that involve proton transfer and radical transfer to tryptophan. A minimal kinetic model is proposed, including a fraction of TyrOH.+ that is stabilized up to the tens of picoseconds timescale, suggesting a potential role of this species as intermediate in biochemical electron transfer reactions.

Direct Observation and Analysis of the Dynamics of the Photoresponsive Transcription Factor GAL4.

Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4-VVD, and DNA using high-speed atomic force microscopy (HS-AFM) is reported. A series of different GAL4-VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter-strand jumping on two double-stranded (ds) DNAs was also observed. Detailed analysis using a long substrate DNA strand containing five GAL4-binding sites revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping to rapidly find specific binding sites, and then stalled to the specific sites to form a stable complex formation. These results suggest the existence of different conformations of the protein to enable sliding and stalling. This single-molecule imaging system with nanoscale resolution provides an insight into the searching mechanism used by DNA-binding proteins.

Improvement of lipid production by the oleaginous yeast Rhodosporidium toruloides through UV mutagenesis.

Oleaginous yeasts are considered a promising alternative lipid source for biodiesel fuel production. In this study, we attempted to improve the lipid productivity of the oleaginous yeast Rhodosporidium toruloides through UV irradiation mutagenesis and selection based on ethanol and H2O2 tolerance or cerulenin, a fatty acid synthetase inhibitor. Glucose consumption, cell growth, and lipid production of mutants were evaluated. The transcription level of genes involved in lipid production was also evaluated in mutants. The ethanol and H2O2 tolerant strain 8766 2-31M and the cerulenin resistant strain 8766 3-11C were generated by UV mutagenesis. The 8766 2-31M mutant showed a higher lipid production rate, and the 8766 3-11C mutant produced a larger amount of lipid and had a higher lipid production rate than the wild type strain. Transcriptional analysis revealed that, similar to the wild type strain, the ACL1 and GND1 genes were expressed at significantly low levels, whereas IDP1 and ME1 were highly expressed. In conclusion, lipid productivity in the oleaginous yeast R. toruloides was successfully improved via UV mutagenesis and selection. The study also identified target genes for improving lipid productivity through gene recombination.

Genetically engineered photoinducible homodimerization system with improved dimer-forming efficiency.

Vivid (VVD) is a photoreceptor derived from Neurospora Crassa that rapidly forms a homodimer in response to blue light. Although VVD has several advantages over other photoreceptors as photoinducible homodimerization system, VVD has a critical limitation in its low dimer-forming efficiency. To overcome this limitation of wild-type VVD, here we conduct site-directed saturation mutagenesis in the homodimer interface of VVD. We have found that the Ile52Cys mutation of VVD (VVD-52C) substantially improves its homodimer-forming efficiency up to 180%. We have demonstrated the utility of VVD-52C for making a light-inducible gene expression system more robust. In addition, using VVD-52C, we have developed photoactivatable caspase-9, which enables optical control of apoptosis of mammalian cells. The present genetically engineered photoinducible homodimerization system can provide a powerful tool to optically control a broad range of molecular processes in the cell.

Ultrasound-assisted generation of ACE-inhibitory peptides from casein hydrolyzed with nanoencapsulated protease.

BACKGROUND: Bioactive peptides generated from milk proteins are eminent ingredients for functional foods and nutraceuticals. Amongst several approaches to release these peptides, hydrolysis of milk proteins with proteolytic enzymes is a promising choice. It is, however, required to inactivate the enzyme after a predetermined time, which leads to impurity of the final product. Immobilization of enzyme molecules can overcome this problem as it simplifies enzyme separation from the reaction mixture. A fungal protease from Aspergillus oryzea was encapsulated within nanoparticles yielded via silicification of polyamidoamine dendrimer template generation 0. It was used to hydrolyze the dominant milk protein (casein) in the absence or presence of sonication. The production of angiotensin converting enzyme (ACE)-inhibitory peptides was monitored during hydrolysis. RESULTS: Sonication did not affect maximum ACE-inhibitory activity but shortened the process sixfold. Ultrafiltration permeate of the centrifugal supernatant of casein solution hydrolyzed during sonication inhibited ACE activity as efficiently as the supernatant obtained from it. CONCLUSION: The protease from Aspergillus oryzea encapsulated within nanospheres is suitable for generation of ACE-inhibitory peptides from casein. The nanoncapsulation procedure is simple, rapid and efficient. This may enable the industrial production of functional products from milk.

Physical interaction between VIVID and white collar complex regulates photoadaptation in Neurospora.

Photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. In Neurospora crassa, VIVID (VVD), a small LOV domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. We report that there is a physical interaction between VVD and the white collar complex (WCC), the primary blue-light photoreceptor and the transcription factor complex that initiates light-regulated transcriptional responses in Neurospora. Using two previously characterized VVD mutants, we show that the level of interaction is correlated with the level of WCC repression in constant light and that even light-insensitive VVD is sufficient partly to regulate photoadaptation in vivo. We provide evidence that a functional GFP-VVD fusion protein accumulates in the nucleus on light induction but that nuclear localization of VVD does not require light. Constitutively expressed VVD alone is sufficient to change the dynamics of photoadaptation. Thus, our results demonstrate a direct molecular connection between two of the most essential light signaling components in Neurospora, VVD and WCC, illuminating a previously uncharacterized process for light-sensitive eukaryotic cells.

The temperature dependence of maltose transport in ale and lager strains of brewer's yeast.

Lager beers are traditionally made at lower temperatures (6-14 degrees C) than ales (15-25 degrees C). At low temperatures, lager strains (Saccharomyces pastorianus) ferment faster than ale strains (Saccharomyces cerevisiae). Two lager and two ale strains had similar maltose transport activities at 20 degrees C, but at 0 degrees C the lager strains had fivefold greater activity. AGT1, MTT1 and MALx1 are major maltose transporter genes. In nine tested lager strains, the AGT1 genes contained premature stop codons. None of five tested ale strains had this defect. All tested lager strains, but no ale strain, contained MTT1 genes. When functional AGT1 from an ale strain was expressed in a lager strain, the resultant maltose transport activity had the high temperature dependence characteristic of ale yeasts. Lager yeast MTT1 and MALx1 genes were expressed in a maltose-negative laboratory strain of S. cerevisiae. The resultant Mtt1 transport activity had low temperature dependence and the Malx1 activity had high temperature dependence. Faster fermentation at low temperature by lager strains than ale strains may result from their different maltose transporters. The loss of Agt1 transporters during the evolution of lager strains may have provided plasma membrane space for the Mtt1 transporters that perform better at a low temperature.

Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues.

Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after approximately 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR *-. Additional mechanisms that may lead to the near disappearance of Trp(*) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Protein release from yeast cells as an evaluation method of physical effects in ultrasonic field.

The release rate of intercellular protein from yeast cells by the ultrasonic action is proposed as a method for evaluating the physical (mechanical) effects of the ultrasonic field. The protein concentration was quantitatively determined using UV absorbance of proteins by spectrophotometry. The detail of the procedures, such as the effects of the origin of yeast cells, pretreatment of the cells, and the wavelengths for spectrophotometric determination of protein content, are examined. The effectiveness of the proposed evaluation method was experimentally demonstrated by changing the irradiation conditions of ultrasound, such as the concentration of yeast cells, temperature, ultrasound power, types of sonicator, and the superposition with the mechanical mixing. The results validate the usefulness of the proposed evaluation method for the quantification of the physical effects of ultrasound fields. Also, the range of cavitational effects of ultrasound sensed by the evaluation procedures were discussed.

Compensatory role for Rad52 during recombinational repair in Ustilago maydis.

A single Rad52-related protein is evident by blast analysis of the Ustilago maydis genome database. Mutants created by disruption of the structural gene exhibited few discernible defects in resistance to UV, ionizing radiation, chemical alkylating or cross-linking agents. No deficiency was noted in spontaneous mutator activity, allelic recombination or meiosis. GFP-Rad51 foci were formed in rad52 cells following DNA damage, but were initially less intense than normal suggesting a possible role for Rad52 in formation of the Rad51 nucleoprotein filament. A search for interacting genes that confer a synthetic fitness phenotype with rad52 after DNA damage by UV irradiation identified the genes for Mph1, Ercc1 and the Rad51 paralogue Rec2. Testing known mutants in recombinational repair revealed an additional interaction with the BRCA2 orthologue Brh2. Suppression of the rec2 mutant's UV sensitivity by overexpressing Brh2 was found to be dependent on Rad52. The results suggest that Rad52 serves in an overlapping, compensatory role with both Rec2 and Brh2 to promote and maintain formation of the Rad51 nucleoprotein filament.

The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation.

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.

4-thio-U cross-linking identifies the active site of the VS ribozyme.

To identify nucleotides in or near the active site, we have used a circularly permuted version of the VS ribozyme capable of cleavage and ligation to incorporate a single photoactive nucleotide analog, 4-thio- uridine, immediately downstream of the scissile bond. Exposure to UV light produced two cross-linked RNAs, in which the 4-thio-uridine was cross-linked to A756 in the 730 loop of helix VI. The cross-links formed only under conditions that support catalytic activity, suggesting that they reflect functionally relevant conformations of the RNA. One of the cross-linked RNAs contains a lariat, indicative of intramolecular cross-linking in the ligated RNA; the other is a branched molecule in which the scissile phosphodiester bond is cleaved, but occupies the same site in the ribozyme-substrate complex. These are the two forms of the RNA expected to be the ground state structures on either side of the transition state. This localization of the active site is consistent with previous mutational, biochemical and biophysical data, and provides direct evidence that the cleavage site in helix I interacts with the 730 loop in helix VI.

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue.

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.

Recessive mutations in SUP35 and SUP45 genes coding for translation release factors affect chromosome stability in Saccharomyces cerevisiae.

Chromosome stability in suppressor mutants for SUP35 and SUP45 genes coding for translation release factors was studied. We obtained spontaneous and UV-induced sup35 or sup45 mutants in a haploid strain disomic for chromosome III and tested the stability of an extra copy of this chromosome. The majority of the mutants showed increased chromosome instability. This phenotype was correlated with an increased sensitivity to the microtubule-poisoning drug benomyl which affects chromosome segregation at anaphase. Our data suggest that termination-translation factors eRF3 and eRF1 control chromosome transmission at mitotic anaphase in Saccharomyces cerevisiae.

[The effect of urea, gamma- and UV-irradiation on physico-chemical characteristics of native and immobilized inulinase]. / Vozdeistvie mocheviny, gamma- i UV--izluchenii na fiziko-khimicheskie svoistva svobodnoi i immobilizovannoi inulazy.

The immobilization of inulinase by ionexchange AB-26 and AB-17-2P has been made by adsorbtion and glutaraldehyde methods. The effect of UV-radiation, carbamide and gamma-rays on the stability of native and immobilized enzyme has been investigated. The stability of inulinase in relation to denaturation agents has been shown to increase with the immobilization of ionexchange. The character of binding with the matrix affects greatly the stability of immobilized enzyme to physical factors.