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Microplastic pollution causes a major concern in the marine environment due to their worldwide distribution, persistence, and adverse effects of these pollutants in the marine ecosystem. Despite its global presence, there is still a lack of information on the effect of microplastics on marine organisms at the molecular level. Herein we demonstrated ingestion and egestion of nano- (0.05 μm) and micro-sized (0.5 and 6 μm) polystyrene microbeads in the marine copepod Paracyclopina nana , and examined molecular responses to exposure to microbeads with in vivo endpoints such as growth rate and fecundity. Also, we proposed an adverse outcome pathway for microplastic exposure that covers molecular and individual levels. This study provides the first insight into the mode of action in terms of microplastic-induced oxidative stress and related signaling pathways in P. nana .

A high-throughput and sensitive screen for the improved expression of gene targets in Saccharomyces cerevisiae is described that is based upon the activity of the luciferase from Gaussia princeps. Using the Unspecific Peroxygenase (UPO) from Agrocybe aegerita ( Aae UPO) as a model protein, improvements in expression, effected through error-prone PCR-based mutation within the signal peptide (SP) domain, can be detected using fusion of the target to Gaussia luciferase encoded downstream of the first folded domain of the Aae UPO protein and luminescent assay of expression supernatants. In this way, previously undiscovered mutations within the SP of Aae UPO that improve expression were revealed, and then applied to the expression of full-length Aae UPO in S. cerevisiae . The system was validated against control expression constructs that were well or poorly expressed and indicated a 13.9-fold improvement in expression for the best mutant over the wild-type SP sequence. It is envisaged that this protocol may be applied generally to the high-throughput detection of improved expression in S. cerevisiae for constructs that are engineered using directed evolution techniques.

Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc’s 1 H, 13 C and 15 N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36–81, 96–145 and containing eight out of the nine helices was determined with a C α -atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.

Secreted reporters are a useful tool in the monitoring of different biological processes in the conditioned medium of cultured cells as well in the blood and urine of experimental animals. Described here is a protocol for detecting the recently established naturally secreted Gaussia luciferase (Gluc) in cultured cells as well as in blood and urine in vivo . Furthermore, the assay for detecting the secreted alkaline phosphatase (SEAP), the most commonly used secreted reporter in serum, is also presented. The Gluc reporter system has several advantages over the SEAP assay, including a much reduced assay time (1–10 min versus 1.5–2 h), 20,000-fold ( in vitro ) or 1,000-fold ( in vivo ) increased sensitivity and a linear range covering over five orders of magnitude of cell number. Additionally, the Gluc signal can be detected in urine and the signal can be localized in animals using in vivo bioluminescence imaging.

Gaussia luciferase (Gluc) is currently known as the smallest naturally secreted luciferase. Due to its small molecular size, high sensitivity, short half-life, and high secretion efficiency, it has become an ideal reporter gene and is widely used in monitoring promoter activity, studying protein-protein interactions, protein localization, high-throughput drug screening, and real-time monitoring of tumor occurrence and development. Although studies have shown that different Gluc mutations exhibit different bioluminescent properties, their mechanisms have not been further investigated. The purpose of this study is to reveal the relationship between the conformational changes of Gluc mutants and their bioluminescent properties through molecular dynamics simulation combined with neural relationship inference (NRI) and Markov models. Our results indicate that, after binding to the luciferin coelenterazine (CTZ), the α-helices of the 109–119 residues of the Gluc Mutant2 (GlucM2, the flash-type mutant) are partially unraveled, while the α-helices of the same part of the Gluc Mutant1 (GlucM1, the glow-type mutant) are clearly formed. The results of Markov flux analysis indicate that the conformational differences between glow-type and flash-type mutants when combined with luciferin substrate CTZ mainly involve the helicity change of α7. The most representative conformation and active pocket distance analysis indicate that compared to the flash-type mutant GlucM2, the glow-type mutant GlucM1 has a higher degree of active site closure and tighter binding. In summary, we provide a theoretical basis for exploring the relationship between the conformational changes of Gluc mutants and their bioluminescent properties, which can serve as a reference for the modification and evolution of luciferases.

Photoproteins have played a major role in advancing our understanding of biological processes. A broader array of biocompatible, nontoxic, and novel reporters can serve to expand this potential. Here we describe the properties of a luciferase from the copepod marine organism Gaussia princeps. It is a monomeric protein composed of 185 aa (19.9 kDa) with a short coding sequence (555 bp) making it suitable for viral vectors. The humanized form of Gaussia luciferase (hGLuc) was efficiently expressed in mammalian cells following delivery by HSV-1 amplicon vectors. It was found to be nontoxic and naturally secreted, with flash bioluminescence characteristics similar to those of other coelenterazine luciferases. hGLuc generated over 1000-fold higher bioluminescent signal intensity from live cells together with their immediate environment and over 100-fold higher intensity from viable cells alone (not including secreted luciferase) or cell lysates, compared to humanized forms of firefly (hFLuc) and Renilla (hRLuc) luciferases expressed under similar conditions. Furthermore, hGLuc showed 200-fold higher signal intensity than hRLuc and intensity comparable to that of hFLuc in vivo under standard imaging conditions. Gaussia luciferase provides a sensitive means of imaging gene delivery and other events in living cells in culture and in vivo, with a unique combination of features including high signal intensity, secretion, and ATP independence, thus being able to report from the cells and their environment in real time.

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

Gaussia Luciferase (GLuc) is a renowned reporter protein that can catalyze the oxidation of coelenterazine (CTZ) and emit a bright light signal. GLuc comprises two consecutive repeats that form the enzyme body and a central putative catalytic cavity. However, deleting the C-terminal repeat only limited reduces the activity (over 30% residual luminescence intensity detectable), despite being a key part of the cavity. How does the remaining GLuc (tGLuc) catalyze CTZ? To address this question, we built a structural model of tGLuc by removing the C-terminal repeat from the resolved structure of intact GLuc, and verified that the cavity-forming component in GLuc remains stable and provides an open-mouth cavity in tGLuc during 500 ns MD simulations in water. Docking simulation and a followed umbrella sampling analysis further revealed that the cavity on tGLuc has a high affinity for CTZ, with a binding energy of up to -114 kJ/mol. Moreover, R76, a validated activity-critical amino acid residue, resides in the cavity and forms a stable hydrogen bond with CTZ. Then, we constructed a cluster model to examine the CTZ oxidation pathway in the cavity using Density Functional Theory (DFT) calculations. The result showed that the pathway consists of four elementary reactions, with the highest Gibbs energy barrier being 65.4 kJ/mol. Both intramolecular electron transfer and the convergence of S1/S0 potential energy surfaces occurred in the last elementary reaction, which was regarded as the reported Chemically-Initiated-Electron-Exchange-Luminescence (CIEEL) reaction. Geometry and wavefunction analysis on the pathway indicated that R76 plays a vital role in CTZ oxidation, which first anchors the environmental oxygen molecule and induces it to form a singlet biradical state, facilitating its attack on CTZ. Subsequently, R76 and the adjacent Q88, positioned near R76 through the tGLuc refolding process, stabilize the transition states and facilitate the emergence of radical electrons on CTZ at the onset of the CIEEL reaction, which contributes to the subsequent intramolecular electron transfer and the production of excited amide product. This study provides a comprehensive explanation of tGLuc’s catalytic mechanism. However, it is important to note that these findings are specific to tGLuc and may not extend to other CTZ-based luciferases, particularly those lacking arginine in their catalytic cavities, which likely operate via distinct mechanisms.

Electron transport system (ETS) function in mitochondria is essential for the aerobic production of energy. Because ETS function requires extensive interactions between mitochondrial and nuclear gene products, coadaptation between mitochondrial and nuclear genomes may evolve within populations. Hybridization between allopatric populations may then expose functional incompatibilities between genomes that have not coevolved. The intertidal copepod Tigriopus californicus has high levels of nucleotide divergence among populations at mitochondrial loci and suffers F2 hybrid breakdown in interpopulation hybrids. We hypothesize that hybridization results in incompatibilities among subunits in ETS enzyme complexes and that these incompatibilities result in diminished mitochondrial function and fitness. To test this hypothesis, we measured fitness, mitochondrial function, and ETS enzyme activity in inbred recombinant hybrid lines of Tigriopus californicus. We found that (1) both fitness and mitochondrial function are reduced in hybrid lines, (2) only those ETS enzymes with both nuclear and mitochondrial subunits show a loss of activity in hybrid lines, and (3) positive relationships exist between ETS enzyme activity and mitochondrial function and between mitochondrial function and fitness. We also present evidence that hybrid lines harboring mitochondrial DNA (mtDNA) and mitochondrial RNA polymerase (mtRPOL) from the same parental source population have higher fitness than those with mtDNA and mtRPOL from different populations, suggesting that mitochondrial gene regulation may play a role in disruption of mitochondrial performance and fitness of hybrids. These results suggest that disruption of coadaptation between nuclear and mitochondrial genes contributes to the phenomenon of hybrid breakdown.