Search Results (36)

Neuronal apoptosis is an early and critical pathological hallmark of many chronic neurodegenerative diseases, often occurring silently long before the appearance of overt clinical symptoms. In this study, we engineered astrocytes utilizing a dual-biomarker recognition synNotch system (dual-synNotch). This system is designed to specifically identify neuronal apoptosis through the ‘AND Gate’ activation mechanism, which is triggered by the simultaneous sensing of the apoptotic signal phosphatidylserine (PS) and the neuronal signal ganglioside Gt1b. Upon detection of these neuronal apoptotic signals, the synNotch receptors are activated, inducing the expression of two key molecules: secreted Gaussia luciferase (GLuc), a highly detectable reporter that can cross the blood–brain barrier (BBB), and brain-derived neurotrophic factor (BDNF), a neuroprotective molecule that promotes neuronal survival by inhibiting apoptosis and enhancing memory and cognitive function. This engineered system effectively converts and amplifies early, imperceptible neuronal apoptotic signals into detectable outputs, enabling convenient in vitro monitoring and diagnosis. Therefore, it represents a promising strategy for the early detection and intervention of neurodegenerative diseases associated with neuronal apoptosis. Full article

Missense mutations are the most prevalent alterations in genetic disorders such as hemophilia A (HA), which results from coagulation factor VIII (FVIII) deficiencies. These mutations disrupt protein biosynthesis, folding, secretion, and function. Current treatments for HA are extremely expensive and inconvenient for patients. Small molecule drugs offer a promising alternative or adjunctive strategy due to their lower cost and ease of administration, enhancing accessibility and patient compliance. By screening drug/chemical libraries with cells stably expressing FVIII–Gaussia luciferase fusion proteins, we identified compounds that enhance FVIII secretion and activity. Among these, suberoylanilide hydroxamic acid (SAHA) improved the secretion and activity of wild-type FVIII and common HA-associated missense mutants, especially mild and moderate ones. SAHA increased FVIII interaction with the endoplasmic reticulum chaperone BiP/GRP78 but not with calreticulin. Lowering cellular BiP levels decreased SAHA-induced FVIII secretion and enhancing BiP expression increased FVIII secretion. SAHA also enhanced secretion and BiP interactions with individual domains of FVIII. In vivo, treating mice with SAHA or a BiP activator boosted endogenous FVIII activity. These findings suggest that SAHA serves as a proteostasis regulator, providing a novel therapeutic approach to improve the secretion and functionality of FVIII missense mutants prone to misfolding. Full article

Antibodies are complex protein structures, and producing them using eukaryotic expression systems presents significant challenges. One frequently overlooked aspect of expression vectors is the nucleotide sequence encoding the signal peptide, which plays a pivotal role in facilitating the secretion of recombinant proteins. This study presents the development of an integrative vector, pVEAL3, for expressing full-length recombinant monoclonal antibodies in mammalian cells. The vector features a distinctive nucleotide sequence that encodes an artificial chimeric signal peptide with the following amino acid sequence: MMRTLILAVLLVYFCATVHC. Additionally, the vector incorporates several regulatory elements to enhance antibody expression, including the Gaussia luciferase signal sequence, internal ribosome entry site (IRES), P2A peptide, and a furin cleavage site. These elements coordinate to regulate the synthesis levels of the antibody chains. The analysis of clones obtained via transfection with the developed vector showed that over 95% of them secreted antibodies at levels significantly higher than those of the control. The immunochemical analysis of the chimeric antibody produced by the CHO-K1-10H10ch cell line confirmed the preservation of its functional activity. Full article

Gaussia luciferase (GLuc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.8 to 7.5-fold compared to wild-type GLuc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in GLuc (designated as GLuc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the GLuc5-secreted reporter in transfected mammalian cells confirmed its superior BL performance across multiple cell lines. These findings suggest that the mutated residues are likely crucial for enhancing BL intensity in GLuc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications. Full article

Background/Objectives: UBL3 (Ubiquitin-like 3) is a protein that plays a crucial role in post-translational modifications, particularly in regulating protein transport within small extracellular vesicles. While previous research has predominantly focused on its interactions with α-synuclein, this study investigates UBL3’s role in Huntington’s disease (HD). HD is characterized by movement disorders and cognitive impairments, with its pathogenesis linked to toxic, polyglutamine (polyQ)-expanded mutant huntingtin fragments (mHTT). However, the mechanisms underlying the interaction between UBL3 and mHTT remain poorly understood. Methods: To elucidate this relationship, we performed hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) on postmortem brain tissue from HD patients. Gaussia princeps-based split-luciferase complementation assay and co-immunoprecipitation were employed to confirm the interaction between UBL3 and mHTT. Additionally, we conducted a HiBiT lytic detection assay to assess the influence of UBL3 on the intracellular sorting of mHTT. Finally, immunocytochemical staining was utilized to validate the colocalization and distribution of these proteins. Results: Our findings revealed UBL3-positive inclusions in the cytoplasm and nuclei of neurons throughout the striatum of HD patients. We discovered that UBL3 colocalizes and interacts with mHTT and modulates its intracellular sorting. Conclusions: These results suggest that UBL3 may play a significant role in the interaction and sorting of mHTT, contributing to the understanding of its potential implications in the pathophysiology of Huntington’s disease. Full article

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. Full article

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson’s disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases. Full article

Essential oils exhibit numerous medicinal properties, including antimicrobial, anti-inflammatory and antioxidant effects. Recent studies also indicate that certain essential oils demonstrate anti-amyloidogenic activity against β-amyloid, the protein implicated in Alzheimer’s disease. To investigate whether the anti-aggregating properties of essential oils extend to α-synuclein, the protein involved in Parkinson’s disease, we constructed and employed a whole-cell biosensor based on the split-luciferase complementation assay. We validated our biosensor by using baicalein, a known inhibitor of α-synuclein aggregation, and subsequently we tested eight essential oils commonly used in food and the hygienic industry. Two of them, citron and sage, along with their primary components, pure linalool (the main constituent in citron essential oil) and pure eucalyptol (1,8-cineole, the main constituent in sage essential oil), were able to reduce α-syn aggregation. These findings suggest that both essential oils and their main constituents could be regarded as potential components in functional foods or incorporated into complementary Parkinson’s disease therapies. Full article

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. Notably, 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression-driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, DoE optimized 14% GelMA/2% OMA, 14% GelMA control, and 16% GelMA (total solid content control) were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, successfully determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion. This strategy allows for rapid analysis of the role(s) of biomaterial composition, stiffness or other cell manipulations on lubricin expression by chondrocytes, which may improve therapeutic strategies for cartilage regeneration. Full article

In vivo imaging has enabled impressive advances in biological research, both preclinical and clinical, and researchers have an arsenal of imaging methods available. Bioluminescence imaging is an advantageous method for in vivo studies that allows for the simple acquisition of images with low background signals. Researchers have increasingly been looking for ways to improve bioluminescent imaging for in vivo applications, which we sought to achieve by developing a bioluminescent probe that could specifically target cells of interest. We chose pancreatic ductal adenocarcinoma (PDAC) as the disease model because it is the most common type of pancreatic cancer and has an extremely low survival rate. We targeted the epidermal growth factor receptor (EGFR), which is frequently overexpressed in pancreatic cancer cells, using an EGFR-specific affibody to selectively identify PDAC cells and delivered a Gaussia luciferase (GLuc) bioluminescent protein for imaging by engineering a fusion protein with both the affibody and the bioluminescent protein. This fusion protein was then complexed with a G5-PAMAM dendrimer nanocarrier. The dendrimer was used to improve the protein stability in vivo and increase signal strength. Our targeted bioluminescent complex had an enhanced uptake into PDAC cells in vitro and localized to PDAC tumors in vivo in pancreatic cancer xenograft mice. The bioluminescent complexes could delineate the tumor shape, identify multiple masses, and locate metastases. Through this work, an EGFR-targeted bioluminescent–dendrimer complex enabled the straightforward identification and imaging of pancreatic cancer cells in vivo in preclinical models. This argues for the targeted nanocarrier-mediated delivery of bioluminescent proteins as a way to improve in vivo bioluminescent imaging. Full article

The multicellular green alga Volvox carteri has emerged as a valuable model organism for investigating various aspects of multicellularity and cellular differentiation, photoreception and phototaxis, cell division, biogenesis of the extracellular matrix and morphogenetic movements. While a range of molecular tools and bioinformatics resources have been made available for exploring these topics, the establishment of cell type-specific promoters in V. carteri has not been achieved so far. Therefore, here, we conducted a thorough screening of transcriptome data from RNA sequencing analyses of V. carteri in order to identify potential cell type-specific promoters. Eventually, we chose two putative strong and cell type-specific promoters, with one exhibiting specific expression in reproductive cells (gonidia), the PCY1 promoter, and the other in somatic cells, the PFP promoter. After cloning both promoter regions, they were introduced upstream of a luciferase reporter gene. By using particle bombardment, the DNA constructs were stably integrated into the genome of V. carteri. The results of the expression analyses, which were conducted at both the transcript and protein levels, demonstrated that the two promoters drive cell type-specific expression in their respective target cell types. Transformants with considerably diverse expression levels of the chimeric genes were identifiable. In conclusion, the screening and analysis of transcriptome data from RNA sequencing allowed for the identification of potential cell type-specific promoters in V. carteri. Reporter gene constructs demonstrated the actual usability of two promoters. The investigated PCY1 and PFP promoters were proven to be potent molecular tools for genetic engineering in V. carteri. Full article

Ubiquitin-like 3 (UBL3) acts as a post-translational modification (PTM) factor and regulates protein sorting into small extracellular vesicles (sEVs). sEVs have been reported as vectors for the pathology propagation of neurodegenerative diseases, such as α-synucleinopathies. Alpha-synuclein (α-syn) has been widely studied for its involvement in α-synucleinopathies. However, it is still unknown whether UBL3 interacts with α-syn, and is influenced by drugs or compounds. In this study, we investigated the interaction between UBL3 and α-syn, and any ensuing possible functional and pathological implications. We found that UBL3 can interact with α-syn by the Gaussia princeps based split luciferase complementation assay in cells and immunoprecipitation, while cysteine residues at its C-terminal, which are considered important as PTM factors for UBL3, were not essential for the interaction. The interaction was upregulated by 1-methyl-4-phenylpyridinium exposure. In drug screen results, the interaction was significantly downregulated by the treatment of osimertinib. These results suggest that UBL3 interacts with α-syn in cells and is significantly downregulated by epidermal growth factor receptor (EGFR) pathway inhibitor osimertinib. Therefore, the UBL3 pathway may be a new therapeutic target for α-synucleinopathies in the future. Full article

Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of M. longa luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues. Because this part of MLuc7 displays high homology with those of other copepod luciferases, our suggestion is that the determined boundaries of the catalytic domain are the same for all known copepod luciferases. The involvement of the flexible C-terminus in the retention of the bioluminescent reaction product in the substrate-binding cavity was confirmed by structural modeling and kinetics study. We also demonstrate that the ML7-N10 mutant (15.4 kDa) with deletion of ten amino acid residues at the N-terminus can be successfully used as a miniature bioluminescent reporter in living cells. Application of a shortened reporter may surely reduce the metabolic load on the host cells and decrease steric and functional interference at its use as a part of hybrid proteins. Full article

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL₁₄-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL₁₄ which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL₁₄ containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES. Full article

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in humans throughout Asia. In the past twenty years, the emergence of the genotype I (GI) JEV as the dominant genotype in Asian countries has raised a significant threat to public health security. However, no clinically approved drug is available for the specific treatment of JEV infection, and the commercial vaccines derived from the genotype III JEV strains merely provided partial protection against the GI JEV. Thus, an easy-to-perform platform in high-throughput is urgently needed for the antiviral drug screening and assessment of neutralizing antibodies specific against the GI JEV. In this study, we established a reverse genetics system for the GI JEV strain (YZ-1) using a homologous recombination strategy. Using this reverse genetic system, a gaussia luciferase (Gluc) expression cassette was inserted into the JEV genome to generate a reporter virus (rGI-Gluc). The reporter virus exhibited similar growth kinetics to the parental virus and remained genetically stable for at least ten passages in vitro. Of note, the bioluminescence signal strength of Gluc in the culture supernatants was well correlated with the viral progenies determined by viral titration. Taking advantage of this reporter virus, we established Gluc readout-based assays for antiviral drug screening and neutralizing antibody detection against the GI JEV. These Gluc readout-based assays exhibited comparable performance to the assays using an actual virus and are less time consuming and are applicable for a high-throughput format. Taken together, we generated a GI JEV reporter virus expressing a Gluc gene that could be a valuable tool for an antiviral drug screening assay and neutralization assay. Full article