Protein Interactions In Cells Research Articles

BAPCP: A comprehensive and user-friendly web tool for identifying biomarkers from protein microarray technologies

Background and Objective:Proteome microarrays are one of the popular high-throughput screening methods for large-scale investigation of protein interactions in cells. These interactions can be measured on protein chips when coupled with fluorescence-labeled probes, helping indicate potential biomarkers or discover drugs. Several computational tools were developed to help analyze the protein chip results. However, existing tools fail to provide a user-friendly interface for biologists and present only one or two data analysis methods suitable for limited experimental designs, restricting the use cases. Methods:In order to facilitate the biomarker examination using protein chips, we implemented a user-friendly and comprehensive web tool called BAPCP (Biomarker Analysis tool for Protein Chip Platforms) in this research to deal with diverse chip data distributions. Results:BAPCP is well integrated with standard chip result files and includes 7 data normalization methods and 7 custom-designed quality control/differential analysis filters for biomarker extraction among experiment groups. Moreover, it can handle cost-efficient chip designs that repeat several blocks/samples within one single slide. Using experiments of the human coronavirus (HCoV) protein microarray and the E. coli proteome chip that helps study the immune response of Kawasaki disease as examples, we demonstrated that BAPCP can accelerate the time-consuming week-long manual biomarker identification process to merely 3 min. Conclusions:The developed BAPCP tool provides substantial analysis support for protein interaction studies and conforms to the necessity of expanding computer usage and exchanging information in bioscience and medicine. The web service of BAPCP is available at https://cosbi.ee.ncku.edu.tw/BAPCP/.

Computational prediction of protein interactions in single cells by proximity sequencing.

Proximity sequencing (Prox-seq) simultaneously measures gene expression, protein expression and protein complexes on single cells. Using information from dual-antibody binding events, Prox-seq infers surface protein dimers at the single-cell level. Prox-seq provides multi-dimensional phenotyping of single cells in high throughput, and was recently used to track the formation of receptor complexes during cell signaling and discovered a novel interaction between CD9 and CD8 in naïve T cells. The distribution of protein abundance can affect identification of protein complexes in a complicated manner in dual-binding assays like Prox-seq. These effects are difficult to explore with experiments, yet important for accurate quantification of protein complexes. Here, we introduce a physical model of Prox-seq and computationally evaluate several different methods for reducing background noise when quantifying protein complexes. Furthermore, we developed an improved method for analysis of Prox-seq data, which resulted in more accurate and robust quantification of protein complexes. Finally, our Prox-seq model offers a simple way to investigate the behavior of Prox-seq data under various biological conditions and guide users toward selecting the best analysis method for their data.

USP46 enhances tamoxifen resistance in breast cancer cells by stabilizing PTBP1 to facilitate glycolysis

Tamoxifen (TAM) is the primary drug for treating estrogen receptor alpha-positive (ER+) breast cancer (BC). However, resistance to TAM can develop in some patients, limiting its therapeutic efficacy. The ubiquitin-specific protease (USP) family has been associated with the development, progression, and drug resistance of various cancers. To explore the role of USPs in TAM resistance in BC, we used qRT-PCR to compare USP expression between TAM-sensitive (MCF-7 and T47D) and TAM-resistant cells (MCF-7R and T47DR). We then modulated USP46 expression and examined its impact on cell proliferation, drug resistance (via CCK-8 and EdU experiments), glycolysis levels (using a glycolysis detection assay), protein interactions (confirmed by co-IP), and protein changes (analyzed through Western blotting). Our findings revealed that USP46 was significantly overexpressed in TAM-resistant BC cells, leading to the inhibition of the ubiquitin degradation of polypyrimidine tract-binding protein 1 (PTBP1). Overexpression of PTBP1 increased the PKM2/PKM1 ratio, promoted glycolysis, and intensified TAM resistance in BC cells. Knockdown of USP46 induced downregulation of PTBP1 protein by promoting its K48-linked ubiquitination, resulting in a decreased PKM2/PKM1 ratio, reduced glycolysis, and heightened TAM sensitivity in BC cells. In conclusion, this study highlights the critical role of the USP46/PTBP1/PKM2 axis in TAM resistance in BC. Targeted therapy against USP46 may represent a promising strategy to improve the prognosis of TAM-resistant patients.

Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

The Interaction between the DOCK7 Protein and the E2 Protein of Classical Swine Fever Virus Is Not Involved with Viral Replication or Pathogenicity.

The classical swine fever virus (CSFV) particle consists of three glycoproteins, all of which have been shown to be important proteins involved in many virus functions, including interaction with several host proteins. One of these proteins, E2, has been shown to be directly involved with adsorption to the host cell and important for virus virulence. Using the yeast two-hybrid system, we have previously shown that CSFV E2 specifically interacts with the (DOCK7) dedicator of cytokinesis, a scaffolding protein. In this report, the interaction between E2 and DOCK7 was evaluated. To confirm the yeast two-hybrid results and to determine that DOCK7 interacts in swine cells with E2, we performed co-immunoprecipitation and proximity ligation assay (PLA). After demonstrating the protein interaction in swine cells, E2 amino acid residues Y65, V283, and T149 were determined to be critical for interaction with Dock7 by using a random mutated library of E2 and a reverse yeast two-hybrid approach. That disruption of these three residues with mutations Y65F, V283D, and T149A abrogated the Dock7-E2 protein interaction. These mutations were then introduced into a recombinant CSFV, E2DOCK7v, by a reverse genomics approach using the highly virulent CSFV Brescia isolate as a backbone. E2DOCKv was shown to have similar growth kinetics in swine primary macrophages and SK6 cell cultures to the parental Brescia strain. Similarly, E2DOCK7v demonstrated a similar level of virulence to the parental Brescia when inoculated in domestic pigs. Animals intranasally inoculated with 105 TCID50 developed a lethal form of clinical disease with virological and hematological kinetics changes indistinguishable from that produced by the parental strain. Therefore, interaction between CSFV E2 and host DOCK7 is not critically involved in the process of virus replication and disease production.

Mechanisms of PML::Rara and GATA2 Protein Interactions in Hematopoietic Cells

Despite excellent outcomes for Acute Promyelocytic Leukemia (APL) patients with combined ATRA and Arsenic Trioxide therapy, the underlying molecular mechanisms by which the PML::RARA fusion oncoprotein initiates APL are not yet clear. We recently found that the ability of PML::RARA to initiate aberrant serial replating (a surrogate for self-renewal) is dependent upon its ability to bind to DNA, since PML::RARA with a missense mutation (C88A) that prevents the binding of RARA to DNA eliminates aberrant replating. Currently, however, there is no consensus regarding the genomic binding sites of PML::RARA in myeloid progenitor cells and APL cells. Also, the transcriptional and epigenetic consequences of PML::RARA expression in primary hematopoietic cells remain unclear. To define the genomic binding sites of PML::RARA, we generated V5 epitope tagged PML::RARA constructs in MSCV-based retroviral vectors, and transduced mouse hematopoietic stem progenitor cells (HSPCs) with retroviruses containing V5-PML::RARA WT, V5-PML::RARA C88A, or an empty vector. Anti-V5 ChIP-seq revealed that PML::RARA binds to 2,677 distinct loci in primary mouse hematopoietic cells, and 2,064 sites in primary human hematopoietic cells. We previously demonstrated that Gata2 expression is induced with PML::RARA, and indeed, we identified strong PML::RARA binding to the distal Gata2 enhancer in both human and mouse HSPCs. Surprisingly, ATAC-seq revealed that the vast majority of PML::RARA binding sites are found in regions that are chromatin-accessible even in WT promyelocytes, suggesting that these regions are “open” prior to PML::RARA expression, perhaps because of the normal binding of “pioneer” transcription factors to these regions in WT cells. Indeed, evaluation of transcription factor binding motifs in the regions occupied by PML::RARA revealed significant enrichment for RARA binding motifs, and also GATA motifs. We therefore performed ChIP-seq on GATA2 in HSPCs, which confirmed enrichment in binding at the same sites. These data suggested that PML::RARA and GATA2 may bind in close proximity at hundreds of loci in the genome. To test this, we performed proximity labeling studies followed by mass spectrometry using PML::RARA WT vs. PML::RARA C88A fused to the biotin ligase TurboID in primary HSPCs. This approach revealed that the interactions of several proteins with PML::RARA (including GATA2) are dependent on the binding of PML::RARA to DNA; this suggests that DNA may in fact act as a scaffold to stabilize these protein:protein interactions ( Figure 1). To determine whether Gata2 may induce increased chromatin accessibility following PML::RARA expression, we performed ATAC-seq following Gata2 or PML::RARA overexpression in WT hematopoietic progenitor cells. 3,157 regions shared increased accessibility with either PML::RARA or Gata2 overexpression. Moreover, at a transcriptional level, we found that 535 (27.4%) of PML::RARA differentially expressed genes were coordinately regulated by Gata2 in WT cells. This suggests that PML::RARA indirectly regulates many of its gene targets via the upregulation of Gata2, and that Gata2 may be required for PML::RARA to initiate its transcriptional program. To test this, we performed serial replating assays using cells where Gata2 was knocked out either before or after PML::RARA expression. Consistent with our previous study, knock out of Gata2 following PML::RARA expression led to increased self-renewal capacity. However, knock out of Gata2 before PML::RARA expression prevented self-renewal. These data suggest a model in which GATA2 “opens” specific chromatin regions in early myeloid cells, which are then accessible to RARA or PML::RARA. The two proteins interact at these sites, tethered by their mutual interactions to their respective DNA binding motifs. PML::RARA binding promotes a pre-leukemic transcriptional program mediated in part by positively regulating Gata2. Gata2 is therefore initially required for PML::RARA to establish its transcriptional program. However, once that program is established, Gata2 paradoxically acts as a tumor suppressor, performing a normal role to limit proliferative excess in hematopoietic stem and progenitor cells.

Applications of genetic code expansion technology in eukaryotes.

Unnatural amino acids (UAAs) have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins. In eukaryotes, genetic code expansion (GCE) enables the incorporation of UAAs and facilitates posttranscriptional modification (PTM), which is not feasible in prokaryotic systems. GCE is also a powerful tool for cell or animal imaging, the monitoring of protein interactions in target cells, drug development, and switch regulation. Therefore, there is keen interest in utilizing GCE in eukaryotic systems. This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.

One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells

BackgroundA commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters.ResultsAs a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable.ConclusionsWe conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

Effects of Glutathionylation on Guanylyltransferase Activity of NS5 N-terminal Capping Domain from Dengue, Japanese Encephalitis, and Zika Viruses.

Glutathionylation is a protein post-translational modification triggered by oxidative stress. The susceptible proteins are modified by the addition of glutathione to specific cysteine residues. Virus infection also induces oxidative stress in the cell, which affects cellular homeostasis. It is not just the cellular proteins but the viral proteins that can also be modified by glutathionylation events, thereby impacting the function of the viral proteins. This study was conducted to identify the effects of modification by glutathionylation on the guanylyltransferase activity of NS5 and identify the cysteine residues modified for the three flavivirus NS5 proteins. The capping domain of NS5 proteins from 3 flaviviruses was cloned and expressed as recombinant proteins. A gel-based assay for guanylyltransferase activity was performed using a GTP analog labeled with the fluorescent dye Cy5 as substrate. The protein modification by glutathionylation was induced by GSSG and evaluated by western blot. The reactive cysteine residues were identified by mass spectrometry. It was found that the three flavivirus proteins behaved in a similar fashion with increasing glutathionylation yielding decreased guanylyltransferase activity. The three proteins also possessed conserved cysteines and they appeared to be modified for all three proteins. The glutathionylation appeared to induce conformational changes that affect enzyme activity. The conformational changes might also create binding sites for host cell protein interactions at later stages of viral propagation with the glutathionylation event, thereby serving as a switch for function change.

Inhibition of Pancreatic Cancer Cells by Different Amyloid Proteins Reveals an Inverse Relationship between Neurodegenerative Diseases and Cancer.

Neurodegenerative diseases and cancers are considered to be two families of diseases caused by completely opposite cell-death mechanisms: the former caused by premature cell death, with the latter due to the increased resistance to cell death. Growing epidemiologic evidence appear to suggest an inverse correlation between neurodegenerative diseases and cancers. However, pathological links, particularly from a protein-cell interaction perspective, between these two families of diseases remains to be proven. Here, a fundamental study investigates the effects of three amyloid proteins of Aβ (associated with AD), hIAPP (associated with T2D), and hCT (associated with MTC) on pancreatic cancer (PANC-1) cells. Collective results demonstrate a general inhibitory activity of all of three amyloid proteins on cancer cell proliferation, but inhibition efficiencies are strongly dependent on amyloid sequence (Aβ, hIAPP, hCT), concentration (IC25, IC50, IC75), and aggregation states (monomers, oligomers). Amyloid proteins exhibit two pathways against cancer cells: amyloid monomer-induced ROS production to inhibit cell growth and amyloid oligomer-induced membrane disruption to kill cells. Collectively, the results demonstrate a general inhibition function of amyloid proteins to induce cancer cell death by preventing cell proliferation, suppressing cell migration, promoting reactive oxygen species production, and disrupting cell membranes.

Functional Analysis of CXCR3 Splicing Variants and Their Ligands Using NanoBiT-Based Molecular Interaction Assays.

CXCR3 regulates leukocyte trafficking, maturation, and various pathophysiological conditions. Alternative splicing generates three CXCR3 isoforms in humans. Previous studies investigated the roles of CXCR3 isoforms, and some biochemical data are not correlated with biological relevance analyses. RT-PCR analyses indicate that most cells express all three splicing variants, suggesting that they may mutually affect the chemokine binding and cellular responses of other splicing variants. Here, we performed an integrative analysis of the functional relations among CXCR3 splicing variants and their chemokine-dependent signaling using NanoBiT live cell protein interaction assays. The results indicated that the CXCR3 N-terminal region affected cell surface expression levels and ligand-dependent activation. CXCR3A was efficiently expressed in the plasma membrane and responded to I-TAC, IP-10, and MIG chemokines. By contrast, CXCR3B had low plasma membrane expression and mediated I-TAC-stimulated cellular responses. CXCR3Alt was rarely expressed on the cell surface and did not mediate any cell responses to the tested chemokines; however, CXCR3Alt negatively affected the plasma membrane expression of CXCR3A and CXCR3B and their chemokine-stimulated cellular responses. Jurkat cells express endogenous CXCR3, and exogenous CXCR3A expression enhanced chemotactic activity in response to I-TAC, IP-10, and MIG. By contrast, exogenous expression of CXCR3B and CXCR3Alt eliminated or reduced the CXCR3A-induced chemotactic activity. The PF-4 chemokine did not activate any CXCR3-mediated cellular responses. NanoBiT technology are useful to integrative studies of CXCR3-mediated cell signaling, and expand our knowledge of the cellular responses mediated by molecular interactions among the splicing variants, including cell surface expression, ligand-dependent receptor activation, and chemotaxis.

STAMP: Spatio-Temporal Association Mapping of Proteins.

We describe a tool, Spatio-Temporal Association Mapping of Proteins (STAMP), for identifying protein interactomes via proximity labeling. For a proof-of-principle study, we use cytidine 5'-triphosphate synthase (CTPS) as an example. CTPS, a metabolic enzyme, forms filamentous structures termed cytoophidia in various tissues. We apply STAMP to a variety of developmental stages and tissues in Drosophila including adult ovaries. Using a cell-specific GAL4 driver, we verify that TurboID can biotinylate the bait protein CTPS, making possible the identification of protein-protein interactions (PPIs) in individual cells. Using the wild-type and mutant CTPS as bait proteins, STAMP results in two distinct sets of proximate proteomes. Our results suggest that STAMP is a feasible tool to catch in vivo PPIs in situ at a defined spatiotemporal resolution.

Fluorescent In Situ Detection of RNA-Protein Interactions in Intact Cells by RNA-PLA.

RNA-protein proximity ligation assay (RNA-PLA) enables the detection of specific RNA-protein interactions in fixed cells. In RNA-PLA, bridging and ligation of a circular DNA template occurs if the target RNA and protein are within 40 nanometers of each other. The resulting circular template is amplified by rolling circle amplification and abundantly recognized by fluorescent antisense DNA oligonucleotides. This strategy therefore enables localization of RNA-protein interactions in situ with high specificity and sensitivity. Here, we describe the use of RNA-PLA to detect interactions between a nuclear viral RNA and a host RNA-binding protein in Epstein-Barr virus (EBV)-infected B cells.

Functional characterization of GATA6 genetic variants associated with mild congenital heart defects

Damaging GATA6 variants can cause moderate congenital heart defects. With the application of next-generation sequencing approaches, various novel GATA6 variants with unknown significance have been identified from a broad spectrum of congenital heart defects. However, functional assessment for distinct GATA6 variants from different severity of congenital heart defects, especially from mild defects, is lacking, which hinders our understanding of the genotype-phenotype correlations and underlying mechanisms.Here, we assessed the functional consequences of nine rare GATA6 variants, which had been implicated as the most significant variants associated with mild congenital heart defects using the largest case and control cohort. We examined the effects of these variants on subcellular localization, transcriptional activity, and protein interactions in 293T or AC16 cells and their ability to rescue heart malformation in gata6 zebrafish mutant.We found that two of these nine variants, Q120X and S424I, significantly decreased transcriptional activity. Additionally, Q120X altered subcellular localization. Consistent with the in vitro results, the in vivo results showed that Q120X and S424I lost their potency to rescue ventricular malformation in gata6 −/− embryos.The results indicated that Q120X and S424I are pathogenic in mild congenital heart defects. Further, the inconsistence of severely impaired Q120X function and mild CHDs phenotype suggested the complexity of the genotype-phenotype correlation between the GATA6 variant and heart phenotype, which may help to inform prenatal genetic counseling and pre-implantation genotyping for congenital heart defects.

Piezoelectric conductive electrospun nanocomposite PCL/Polyaniline/Barium Titanate scaffold for tissue engineering applications

Recent trends in tissue engineering technology have switched to electrical potentials generated through bioactive scaffolds regarding their appropriate effects on cell behaviors. Preparing a piezo-electrical stimuli scaffold with high electrical conductivity for bone and cartilage tissue regeneration is the ultimate goal of the present study. Here, Barium Titanate nanoparticles (BaTiO3 NPs) were used as piezoelectric material and highly conductive binary doped Polyaniline nanoparticles (PANI NPs) were synthesized by oxidative polymerization. Polycaprolactone (PCL) was applied as carrier substrate polymer and conductive spun nanofibrous scaffolds of PCL/PANI composites were prepared in two different amounts of PANI (3 and 5 wt.%). The conductivity of PCL/PANI nanofibers has been analyzed by standard four probes test. Based on the obtained results, the PCL/PANI5 (with 5 wt.% PANI) was selected due to the superior electrical conductivity of 8.06 × 10–4 s cm - 1. Moreover, the piezoelectric nanofibrous scaffolds of PCL/BT composite were electrospun in three different amounts of BT (20, 30, and 40 wt.%). To investigate the synergic effect of conductive PANI and piezoelectric BT, ternary nanocomposite scaffolds of PCL/PANI/BT were prepared using the dual jet electrospinning technique. The piezoelectric properties have been analyzed by determining the produced voltage. The morphological assessment, contact angle, mechanical test, and MTT assay have been conducted to evaluate other properties including biocompatibility of nanofibrous scaffolds. The PCL/PANI5/BT40 composite resulted in an unprecedented voltage of 1.9 Volt. SEM results confirm that BT NPs have been distributed and embedded inside PCL fibers quite appropriately. Also, the chosen scaffolds were homogeneously intertwined and possessed an average fiber diameter of 288 ± 180 nm, and a contact angle of 92 ± 7°, making it a desirable surface for cell attachment and protein interactions. Moreover, Young’s modulus, ultimate tensile stress, and elongation were obtained as 11 ± 1 MPa, 5 ± 0.6 MPa, and 109 ± 15% respectively. Obtained results assert the novel potential of piezo-electrical stimuli conductive nanocomposite scaffold for tissue engineering applications.

An RNA G-Quadruplex Structure within the ADAR 5'UTR Interacts with DHX36 Helicase to Regulate Translation.

RNA G-quadruplex (rG4) structures in the 5' untranslated region (5'UTR) play crucial roles in fundamental cellular processes. ADAR is an important enzyme that binds to double-strand RNA and accounts for the conversion of Adenosine to Inosine in RNA editing. However, so far there is no report on the formation and regulatory role of rG4 on ADAR expression. Here, we identify and characterize a thermostable rG4 structure within the 5'UTR of the ADAR1 mRNA and demonstrate its formation and inhibitory role on translation in reporter gene and native gene constructs. We reveal rG4-specific helicase DHX36 interacts with this rG4 in vitro and in cells under knockdown and knockout conditions by GTFH (G-quadruplex-triggered fluorogenic hybridization) probes and modulates translation in an rG4-dependent manner. Our results further substantiate the rG4 structure-DHX36 protein interaction in cells and highlight rG4 to be a key player in controlling ADAR1 translation.

An RNA G‐Quadruplex Structure within the ADAR 5′UTR Interacts with DHX36 Helicase to Regulate Translation

AbstractRNA G‐quadruplex (rG4) structures in the 5′ untranslated region (5′UTR) play crucial roles in fundamental cellular processes. ADAR is an important enzyme that binds to double‐strand RNA and accounts for the conversion of Adenosine to Inosine in RNA editing. However, so far there is no report on the formation and regulatory role of rG4 on ADAR expression. Here, we identify and characterize a thermostable rG4 structure within the 5′UTR of the ADAR1 mRNA and demonstrate its formation and inhibitory role on translation in reporter gene and native gene constructs. We reveal rG4‐specific helicase DHX36 interacts with this rG4 in vitro and in cells under knockdown and knockout conditions by GTFH (G‐quadruplex‐triggered fluorogenic hybridization) probes and modulates translation in an rG4‐dependent manner. Our results further substantiate the rG4 structure‐DHX36 protein interaction in cells and highlight rG4 to be a key player in controlling ADAR1 translation.

Speciation Analysis Highlights the Interactions of Auranofin with the Cytoskeleton Proteins of Lung Cancer Cells.

Two types of lung cells (epithelial cancer lung cells, A-549 and lung fibroblasts MRC-5) were exposed to the clinically established gold drug auranofin at concentrations close to the half-maximal inhibitory drug concentrations (IC50). Collected cells were subjected to speciation analysis using inductively coupled plasma mass spectrometry (ICP-MS). Auranofin showed better affinity toward proteins than DNA, RNA, and hydrophilic small molecular weight compounds. It can bind to proteins that vary in size (~20 kDa, ~75 kDa, and ≥200 kDa) and pI. However, the possibility of dimerization and protein–protein complex formation should also be taken into account. µRPLC/CZE-ESI-MS/MS studies on trypsinized proteins allowed the indication of 76 peptides for which signal intensity was influenced by auranofin presence in cells. Based on it, identity was proposed for 20 proteins. Except for thioredoxin reductase (TrxR), which is directly targeted by gold complex, the proteins were found to be transformed. Five indicated proteins: myosin, plectin, talin, two annexins, and kinase M3K5, are responsible for cell–cell, cell–protein interactions, and cell motility. A wound healing test confirmed their regulation by auranofin as cell migration decreased by 40% while the cell cycle was not interrupted.

An investigation of bacteriocin nisin anti-cancer effects and FZD7 protein interactions in liver cancer cells

The bacteriocin, nisin, produced by Lactococcus and Streptococcus species during fermentation, is widely used for bio preservatives in a wide variety of foods. Liver cancer has a high mortality rate and is the fourth leading cause of cancer-related deaths worldwide. Recently, researchers have shown the anti-cancer effects of nisin through in vitro and in vivo studies. This study aimed to investigate the effect of nisin on liver cancer cell lines, which represented two subgroups of the disease model. Nisin exhibited significant growth inhibition and apoptosis in both cell lines, HuH-7, and SNU182. Drug resistance is the main problem in liver cancer and the epithelial-to-mesenchymal transition has a role in the development of drug resistance in hepatocellular carcinoma. The expression of EMT transcription factors ZEB1, SNAI1, and TWIST1 were analyzed depending on nisin treatment, TWIST1 expression was down-regulated after nisin treatment compared to the untreated SNU182 and HuH-7 cell lines. Besides, due to the reported correlation between the overexpression of Frizzled (FZD) proteins, specifically FZD7, in primary hepatocellular carcinomas (HCCs), molecular docking was assessed for Nisin A in the binding site of FZD7. Results confirmed that Nisin A was able to form important hydrogen bonding with key residues. This research not only determined the role of nisin in different liver cancer cell models but it also provided the first result of FZD7 and nisin interaction.

Ubiquitination and Ubiquitin-Like Modifications as Mediators of Alternative Pre-mRNA Splicing in Arabidopsis thaliana.

Alternative splicing (AS) is a common post-transcriptional regulatory process in eukaryotes. AS has an irreplaceable role during plant development and in response to environmental stress as it evokes differential expression of downstream genes or splicing factors (e.g., serine/arginine-rich proteins). Numerous studies have reported that loss of AS capacity leads to defects in plant growth and development, and induction of stress-sensitive phenotypes. A role for post-translational modification (PTM) of AS components has emerged in recent years. These modifications are capable of regulating the activity, stability, localization, interaction, and folding of spliceosomal proteins in human cells and yeast, indicating that PTMs represent another layer of AS regulation. In this review, we summarize the recent reports concerning ubiquitin and ubiquitin-like modification of spliceosome components and analyze the relationship between spliceosome and the ubiquitin/26S proteasome pathway in plants. Based on the totality of the evidence presented, we further speculate on the roles of protein ubiquitination mediated AS in plant development and environmental response.