Split Luciferase Complementation Assay Research Articles

UBL3 Interacts with PolyQ-Expanded Huntingtin Fragments and Modifies Their Intracellular Sorting.

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. 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. 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. 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.

Subcellular imaging of MGAT1/MGAT2 homo- and heteromers in living cells using bioluminescence microscopy

Protein-protein interactions (PPIs) play fundamental roles in many biological processes including the functioning of glycosylation machineries present in the endoplasmic reticulum (ER) and Golgi apparatus of mammalian cells. For the last couple of years, we have been successfully employing the most advanced version of the split luciferase complementation assay, termed NanoBiT, to demonstrate PPIs between solute carrier 35 (SLC35) family members with nucleotide sugar transporting activity and functionally related glycosyltransferases. NanoBiT has several unmatched advantages as compared with other strategies for studying PPIs. Firstly, the tendency of the free luciferase fragments to spontaneously associate is strongly reduced. As a consequence, the fragments of the reconstituted luciferase may dissociate upon the disruption of the PPI of interest. Secondly, the recombinant fusion proteins are expressed at low (near-endogenous) levels. Both of these features significantly minimize the possibility of obtaining false positive results. In this study we pushed the boundaries of this already powerful technique even further by coupling it with bioluminescence imaging of PPIs. Specifically, we visualized homo- and heterologous complexes formed by MGAT1 and MGAT2 glycosylation enzymes tagged with NanoBiT fragments and demonstrated ER-to-Golgi transitions between enzyme homo- and heteromers.

Crosstalk among WEE1 Kinase, AKT, and GSK3 in Nav1.2 Channelosome Regulation.

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.

Alpha-Synuclein Interaction with UBL3 Is Upregulated by Microsomal Glutathione S-Transferase 3, Leading to Increased Extracellular Transport of the Alpha-Synuclein under Oxidative Stress.

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.

Insights into Phakopsora pachyrhizi Effector-Effector Interactions.

The multifaceted role of pathogen-encoded effectors in plant-pathogen interactions is complex and not fully understood. Effectors operate within intricate host environments, interacting with host proteins and other effectors to modulate virulence. The complex interplay between effectors raises the concept of metaeffectors, wherein some effectors regulate the activity of others. While previous research has demonstrated the importance of effector repertoires in pathogen virulence, only a limited number of studies have investigated the interactions between these effectors. This study explores the interactions among Phakopsora pachyrhizi effector candidates (PpECs). P.pachyrhizi haustorial transcriptome analysis identified a collection of predicted PpECs. Among these, PpEC23 was found to interact with PpEC48, prompting further exploration into their potential interaction with other effectors. Here, we utilized a yeast two-hybrid screen to explore protein-protein interactions between PpECs. A split-luciferase complementation assay also demonstrated that these interactions could occur within soybean cells. Interestingly, PpEC48 displayed the ability to interact with several small cysteine-rich proteins (SCRPs), suggesting its affinity for this specific class of effectors. We show that these interactions involve a histidine-rich domain within PpEC48, emphasizing the significance of structural motifs in mediating effector interactions. The unique nature of PpEC48, showing no sequence matches in other organisms, suggests its relatively recent evolution and potential orphan gene status. Our work reveals insights into the intricate network of interactions among P. pachyrhizi effector-effector interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Mass spectrometry and split luciferase complementation assays reveal the MecA protein interactome of Streptococcus mutans.

MecA is a highly conserved adaptor protein encoded by prokaryotes from the Bacillota phylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiont Streptococcus mutans likely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactions in vivo. In addition, we further develop a new application of SLCA to support in vivo measurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as-yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of the MecA regulatory function still has yet to be discovered.IMPORTANCEDespite multiple decades of study, the regulatory mechanism and function of MecA have remained largely a mystery. The current study provides the first detailed roadmap to investigate these functions in other medically significant bacteria. Furthermore, this study developed new genetic approaches to assay prokaryotic protein-protein interactions via the split luciferase complementation assay (SLCA). SLCA technology is commonly employed in eukaryotic genetic research but has not yet been established for studies of bacterial protein-protein interactions. The SLCA protein binding affinity assay described here is a new technological advance exclusive to the current study and has not been reported elsewhere.

Assessment of the Anti-Amyloidogenic Properties of Essential Oils and Their Constituents in Cells Using a Whole-Cell Recombinant Biosensor.

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.

TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria

BackgroundExcess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes.MethodsThe split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing.ResultsExploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1–JAK2–FGF14–Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection.ConclusionsFGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

The GmCYP2-GmHAL3 module regulates salt tolerance in soybean seedlings

Soybean (Glycine max L. Merr) production is severely affected by soil salinization as an important crop. The discovery of salt stress-responsive genes is essential for soybean breeding. To our knowledge, cyclophilins (CYPs) play important roles in salt stresses in crops such as rice and cotton, except in soybean. This study cloned GmCYP2 from soybean salt-tolerant cultivar Suxie No. 1 and obtained ten stable transgenic soybean lines overexpressing GmCYP2 by Agrobacterium rhizogenes-mediated transformation. Two of the higher-expressing transgenic lines (OE1 and OE2) were used to compare salt tolerance with the wild type (WT). Under the salt stress, the chlorophyll content, the maximum efficiency of PSII photochemistry (Fv/Fm) and actual photochemical quantum yield [Y(II)] of OE1 and OE2 lines were significantly higher than those of the WT. Meanwhile, the accumulation of malondialdehyde (MDA) in the OE1 and OE2 lines was significantly lower than that in the WT. In addition, the Na+/K+ ratio was also significantly lower than that of the WT in leaves and roots. The results also confirmed that the salt tolerance of GmCYP2-silencing soybean seedlings obtained by virus-induced gene silencing (VIGS) was reduced compared to the control. Subcellular localization showed that GmCYP2 was expressed in the plasma membrane and nucleus of Nicotiana benthamiana. RNA-seq results demonstrated that GmCYP2 is involved in key regulators of the ABA and Ca2+ signaling pathway. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and split-luciferase complementation assay demonstrated that GmCYP2 protein interacts with salt-associated halotolerance 3 (HAL3) protein. These results suggest that the GmCYP2-GmHAL3 module enhances salt tolerance of soybean seedlings by maintaining the Na+/K+ ratio and good photosynthetic state and it may be involved in the ABA and Ca2+ signaling pathway.

BOD1L Facilitates Chromatin Binding of SETD1A and Promotes Leukemia Cell Growth

The H3K4 methyltransferase SETD1A plays an essential role in leukemia through a non-catalytic activity which regulates transcriptional pause release of RNA polymerase II. SETD1A localizes to the transcriptional start site of numerous actively transcribed genes, but primarily regulates the expression of a restricted set of genes within the DNA damage response as well as heme biosynthesis pathway. However, the roles of upstream regulators or essential components of SETD1A chromatin binding remain unclear. Using the DepMap database and a CRISPR-tiling screen method, we found that BOD1L is the most correlated co-dependency with SETD1A in human cancer cell lines and that the Shg1 domain of BOD1L plays an indispensable role in human MOLM-13 leukemia cell line. BOD1L knockout increases apoptosis and reduces cell cycle. The strong BOD1L dependency in AML cells was also confirmed using a mouse MLL-r leukemia model in vitro and in vivo. SETD1A catalytic domain mutant AML cells still responded to the BOD1L sgRNAs that suggests the role of BOD1L is independent from the SETD1A catalytic function. Importantly, BOD1L knockout mimics the transcriptional profiles of SETD1A knockout cells and reduces the expression of genes in DNA damage response and heme biosynthesis. BOD1L loss immediately reduces SETD1A distribution on chromatin and increased the NELFE and RNA polymerase II signals at transcriptional start sites. These results show that BOD1L is an essential component of the non-catalytic SETD1A function in transcriptional pause release. We also performed an immunoprecipitation assay using tagged constructs to examine the interaction between BOD1L and SETD1A. Our data indicate that SETD1A associates with BOD1L through the FLOS domain, especially in the C-terminal region. In addition, we found that the loop structure in the Shg1 domain of BOD1L is associated with the nonenzymatic domain of SETD1A. A split luciferase complementation assay reveals that the BOD1L Shg1 domain and SETD1A FLOS domain interact. To demonstrate the role of BOD1L chromatin interaction, we enforced localization of the Shg1 domain fragment at a silent gene locus and it successfully induced ectopic SETD1A recruitment to chromatin and formation of the COMPASS complex. However, we observed neither induced H3K4me3 modification nor RNA polymerase II recruitment. Taken together, while the BOD1L-SETD1A complex is not sufficient to act as a pioneer complex for active transcription, our study identified that BOD1L facilitates chromatin binding of SETD1A through the Shg1-FLOS domain interaction and then regulates transcriptional pause release, and that this interaction is a potential therapeutic opportunity for SETD1A-targeting therapy.

The Split-Luciferase Complementation Assay to Detect and Quantify Protein-Protein Interactions in Planta.

Protein-protein interactions play a critical role in plant viral infection and defense responses against pathogens. This protocol provides a detailed and reliable methodology for investigating protein-protein interactions using a luciferase-based complementation assay that includes easy luminescence-based normalization within a single plate. The protocol includes step-by-step procedures, reagent lists, and considerations for data interpretation, ensuring robust and reproducible results. By following this protocol, researchers can advance on understanding of the crucial role of protein-protein interactions in plant viral infection and defense responses to other pathogen attacks.

UBL3 Interaction with α-Synuclein Is Downregulated by Silencing MGST3.

Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that plays a crucial role in sorting proteins into small extracellular vesicles. Aggregations of alpha-synuclein (α-syn) are associated with the pathology of neurodegenerative diseases such as Parkinson's disease. Recently, the interaction between UBL3 and α-syn was discovered, with potential implications in clearing excess α-syn from neurons and its role in disease spread. However, the regulator that can mediate the interaction between UBL3 and α-syn remains unclear. In this study, using the split gaussian luciferase complementation assay and RNA interference technology, we identified that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1, and ATG3 were involved in downregulating the interaction between UBL3 and α-syn. Notably, silencing MGST3 had the most significant impact. Immunocytochemistry staining confirmed the impact of MGST3 silencing on the co-localization of UBL3 and α-syn in cells. MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We induced oxidative stress with hydrogen peroxide, observing its effect on the UBL3-α-syn interaction, and showing that 800 µM of H2O2 downregulated this interaction. In conclusion, silencing MGST3 downregulates the interaction between UBL3 and α-syn.

The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis.

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor Camellia sinensis Senescnece 40 (CsS40) as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase (TCS1) promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of CsS40 in 'Fuding Dabai' developing leaves was opposite to that of TCS1; and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels, respectively. The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of 'Fuding Dabai'. Furthermore, overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors. Taken together, we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar 'Fuding Dabai'. The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.

UBL3 Interacts with Alpha-Synuclein in Cells and the Interaction Is Downregulated by the EGFR Pathway Inhibitor Osimertinib.

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.

Arabidopsis calmodulin-like proteins CML13 and CML14 interact with proteins that have IQ domains.

In response to Ca2+ signals, the evolutionarily-conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM-like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as 'bait' in a yeast two-hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin-binding transcriptional activators (CAMTAs)and myosins, all of which possess tandem isoleucine-glutamine (IQ) structural domains. Using the split-luciferase complementation assay in planta and the yeast 2-hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non-IQ, CaM-binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic-nucleotide gated channel-20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ-protein and found that only CaM, CML13and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+ . Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein-tagged versions of CaM, CML13and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co-expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins.

Near-Infrared Luciferase Complementation Assay with Enhanced Bioluminescence for Studying Protein-Protein Interactions and Drug Evaluation Under Physiological Conditions.

Identification of protein-protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by ∼3.79-fold and ∼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2.

Development and characterization of an inducible assay system to measure Zika virus capsid interactions.

The global spread of the mosquito-borne Zika virus (ZIKV) infection and its complications including Guillain-Barré syndrome and fetus microcephaly in 2015 have made ZIKV as a significant public health threat. The capsid protein plays crucial roles in ZIKV replication and thus represents an attractive therapeutic target. However, inhibitors of ZIKV capsid assembly have not been rigorously identified due to the lack of a target-based screening system. In this study, we developed a novel ZIKV capsid interaction method based on a split-luciferase complementation assay, which can be used to measure and quantify ZIKV capsid-capsid (C-C) interaction by the restored luciferase signal when capsid proteins interact with each other. Furthermore, a Tet-on inducible stable cell line was generated to screen inhibitors of capsid dimerization. By using of this system, peptides (Pep.15-24 in the N-terminal region of ZIKV capsid protein and Pep.44-58 in the α2 helix of ZIKV capsid protein) were identified to inhibit ZIKV C-C interaction. Overall, this study developed a novel inducible assay system to measure ZIKV capsid interaction and identify ZIKV capsid multimerization inhibitors, which will be applied for future discovery of ZIKV assembly inhibitors.

Identification of NLRP3PYD Homo-Oligomerization Inhibitors with Anti-Inflammatory Activity.

Inflammasomes are multiprotein complexes that represent critical elements of the inflammatory response. The dysregulation of the best-characterized complex, the NLRP3 inflammasome, has been linked to the pathogenesis of diseases such as multiple sclerosis, type 2 diabetes mellitus, Alzheimer’s disease, and cancer. While there exist molecular inhibitors specific for the various components of inflammasome complexes, no currently reported inhibitors specifically target NLRP3PYD homo-oligomerization. In the present study, we describe the identification of QM380 and QM381 as NLRP3PYD homo-oligomerization inhibitors after screening small molecules from the MyriaScreen library using a split-luciferase complementation assay. Our results demonstrate that these NLRP3PYD inhibitors interfere with ASC speck formation, inhibit pro-inflammatory cytokine IL1-β release, and decrease pyroptotic cell death. We employed spectroscopic techniques and computational docking analyses with QM380 and QM381 and the PYD domain to confirm the experimental results and predict possible mechanisms underlying the inhibition of NLRP3PYD homo-interactions.

Split-Luciferase Complementation for Analysis of Virus-Host Protein Interactions.

Productive viral infection entails highly regulated and sequential protein-protein interactions between viral factors and between virus and host factors. Deciphering such interactions is of paramount importance for a better understanding of virus infection cycles and the development of new strategies for virus prevention and control. In this protocol, we describe a split-luciferase complementation (SLC ) assay for the detection of protein-protein interaction in Nicotiana benthamiana leaves following agroinfiltration-mediated transient protein expression. In this assay, the firefly luciferase protein is divided into two halves, each expressed as a fusion to a prey or bait protein, respectively. Interaction of the two candidate proteins brings the two otherwise nonfunctional halves into close proximity to restore the luciferase activity, which catalyzes the substrate D-luciferin to emit luminescence. The SLC assay allows for noninvasive, quantitative measurement of dynamic protein interactions in living cells within their native cellular compartments.

Split-luciferase complementary assay of NLRP3 PYD-PYD interaction indicates inflammasome formation during inflammation

The NLRP3 inflammasome is a key macromolecular complex of the innate immune system that activates the inflammatory signalling cascade in response to a wide range of stimuli. Structural studies have shown that the intracellular cytosolic receptor NLRP3 oligomerizes upon stimulation and serves as a scaffold to form the ASC filaments necessary for procaspase-1 activation. Despite the abundant structural evidences on NLRP3 inflammasome, the interactions of the NLRP3 Pyrin domain and its functional relevance are poorly understood. In this study, the split luciferase complementation assay is used as an alternative approach to investigate NLRP3PYD-NLRP3PYD interactions during inflammasome formation. Since the homotypic NLRP3 interaction is mainly based on electrostatic interactions, a phosphomimetic residue (S5) at the interface of the NLRP3PYDs interactions has been mutated to show a disruptive effect on luciferase activity. According to the results presented, the designed biosensor was able to monitor the NLRP3PYD-NLRP3PYD interaction in vitro. The current reporter assay not only provides a specific NLRP3PYD-NLRP3PYD assay to study the PYD-PYD interaction in vitro, but also provides a suitable system for screening chemicals and drugs to identify activators and inhibitors of NLRP3.