Revealing protein-protein interactions at the transcriptome scale by sequencing.

We describe PROPER-seq (protein-protein interaction sequencing) to map protein-protein interactions (PPIs) en masse. PROPER-seq first converts transcriptomes of input cells into RNA-barcoded protein libraries, in which all interacting protein pairs are captured through nucleotide barcode ligation, recorded as chimeric DNA sequences, and decoded at once by sequencing and mapping. We applied PROPER-seq to human embryonic kidney cells, T lymphocytes, and endothelial cells and identified 210,518 human PPIs (collected in the PROPER v.1.0 database). Among these, 1,365 and 2,480 PPIs are supported by published co-immunoprecipitation (coIP) and affinity purification-mass spectrometry (AP-MS) data, 17,638 PPIs are predicted by the prePPI algorithm without previous experimental validation, and 100 PPIs overlap human synthetic lethal gene pairs. In addition, four previously uncharacterized interaction partners with poly(ADP-ribose) polymerase 1 (PARP1) (a critical protein in DNA repair) known as XPO1, MATR3, IPO5, and LEO1 are validated in vivo. PROPER-seq presents a time-effective technology to map PPIs at the transcriptome scale, and PROPER v.1.0 provides a rich resource for studying PPIs.

XLG2 and CORI3 function additively to regulate plant defense against the necrotrophic pathogen Sclerotinia sclerotiorum.

The membrane-bound heterotrimeric G-proteins in plants play a crucial role in defending against a broad range of pathogens. This study emphasizes the significance of Extra-large Gα protein 2 (XLG2), a plant-specific G-protein, in mediating the plant response to Sclerotinia sclerotiorum, which infects over 600 plant species worldwide. Our analysis of Arabidopsis G-protein mutants showed that loss of XLG2 function increased susceptibility to S. sclerotiorum, accompanied by compromised accumulation of jasmonic acid (JA) during pathogen infection. Overexpression of the XLG2 gene in xlg2 mutant plants resulted in higher resistance and increased JA accumulation during S. sclerotiorum infection. Co-immunoprecipitation (co-IP) analysis on S. sclerotiorum infected Col-0 samples, using two different approaches, identified 201 XLG2-interacting proteins. The identified JA-biosynthetic and JA-responsive proteins had compromised transcript expression in the xlg2 mutant during pathogen infection. XLG2 was found to interact physically with a JA-responsive protein, Coronatine induced 1 (CORI3) in Co-IP, and confirmed using split firefly luciferase complementation and bimolecular fluorescent complementation assays. Additionally, genetic analysis revealed an additive effect of XLG2 and CORI3 on resistance against S. sclerotiorum, JA accumulation, and expression of the defense marker genes. Overall, our study reveals two independent pathways involving XLG2 and CORI3 in contributing resistance against S. sclerotiorum.

Extraction method combining saponin and trehalose useful for analyzing fragile intermolecular association.

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.

A novel fluorescent protein pair facilitates FLIM-FRET analysis of plant immune receptor interaction under native conditions.

Elucidating protein-protein interactions is crucial for our understanding of molecular processes within living organisms. Microscopy-based techniques can detect protein-protein interactions in vivo at the single-cell level and provide information on their subcellular location. Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) is one of the most robust imaging approaches, but it is still very challenging to apply this method to proteins which are expressed under native conditions. Here we describe a novel combination of fluorescence proteins (FPs), mCitrine and mScarlet-I, which is ideally suited for FLIM-FRET studies of low abundance proteins expressed from their native promoters in stably transformed plants. The donor mCitrine displays excellent brightness in planta, near-mono-exponential fluorescence decay, and a comparatively long fluorescence lifetime. Moreover, the FRET pair has a good spectral overlap and a large Förster radius. This allowed us to detect constitutive as well as ligand-induced interaction of the Arabidopsis chitin receptor components CERK1 and LYK5 in a set of proof-of-principle experiments. Due to the good brightness of the acceptor mScarlet-I, the FP combination can be readily utilized for co-localization studies. The FP pair is also suitable for co-immunoprecipitation experiments and western blotting, facilitating a multi-method approach for studying and confirming protein-protein interactions.

Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

BACKGROUND: Tissue factor (TF) activity is stringently regulated through processes termed encryption. Post-translational modification of TF and its interactions with various protein and lipid moieties allows for a multi-step de-encryption of TF and procoagulant activation. Membrane-associated guanylate kinase-with inverted configuration (MAGI) proteins are known to regulate the localisation and activity of a number of proteins including cell-surface receptors. METHODS: The interaction of TF with MAGI1 protein was examined as a means of regulating TF activity. MDA-MB-231 cell line was used which express TF and MAGI1, and respond well to protease activated receptor (PAR)2 activation. Proximity ligation assay (PLA), co-immunoprecipitation and pull-down experiments were used to examine the interaction of TF with MAGI1-3 proteins and to investigate the influence of PAR2 activation. Furthermore, by cloning and expressing the PDZ domains from MAGI1, the TF-binding domain was identified. The ability of the recombinant PDZ domains to act as competitors for MAGI1, allowing the induction of TF procoagulant and signalling activity was then examined. RESULTS: PLA and fluorescence microscopic analysis indicated that TF predominantly associates with MAGI1 and less with MAGI2 and MAGI3 proteins. The interaction of TF with MAGI1 was also demonstrated by both co-immunoprecipitation of TF with MAGI1, and co-immunoprecipitation of MAGI1 with TF. Moreover, activation of PAR2 resulted in reduction in the association of these two proteins. Pull-down assays using TF-cytoplasmic domain peptides indicated that the phosphorylation of Ser253 within TF prevents its association with MAGI1. Additionally, the five HA-tagged PDZ domains of MAGI1 were overexpressed separately, and the putative TF-binding domain was identified as PDZ1 domain. Expression of this PDZ domain in cells significantly augmented the TF activity measured both as thrombin-generation and also TF-mediated proliferative signalling. CONCLUSIONS: Our data indicate a stabilising interaction between TF and the PDZ-1 domain of MAGI1 and demonstrate that the activation of PAR2 disrupts this interaction. The release of TF from MAGI1 appears to be an initial step in TF de-encryption, associated with increased TF-mediated procoagulant and signalling activities. This mechanism is also likely to lead to further interactions and modifications leading to further enhancement of procoagulant activity, or the release of TF.

Deciphering MMP9's dual role in regulating SOD3 through protein-protein interactions.

Although the collagenase enzyme activity of matrix metalloproteinase-9 (MMP9) is well-documented, its non-enzymatic functions remain less understood. The interaction between intracellular superoxide dismutase-1 (SOD1) and MMP9 is known, with SOD1 suppressing MMP9. However, the mechanism by which MMP9, a secretory protein, influences the extracellular antioxidant superoxide dismutase-3 (SOD3) is not yet clear. To explore MMP9's regulatory impact on SOD3, we employed human embryonic kidney-293 cells, transfecting them with MMP9 overexpresssion and catalytic-site mutant plasmids. Additionally, MMP9 overexpressing cells were treated with an MMP9 activator and inhibitor. Analyses of both cell lysates and culture medium provided insights into MMP9's intracellular and extracellular regulatory roles. In-silico analysis and experimental approaches like proximal ligation assay and co-immunoprecipitation were utilized to delineate the protein-protein interactions between MMP9 and SOD3. Our findings indicate that activated MMP9 enhances SOD3 levels, a regulation not hindered by MMP9 inhibitors. Intriguingly, catalytically inactive MMP9 appeared to reduce SOD3 levels, likely due to MMP9's binding with SOD3, leading to their proteolytic degradation. This MMP9 influence on SOD3 was consistent in both intracellular and extracellular environments, suggesting a parallel in MMP9-SOD3 interactions across these domains. Ultimately, this study unveils a novel interaction between MMP9 and SOD3, highlighting the unique regulatory role of catalytically inactive MMP9 in diminishing SOD3 levels, contrasting its usual upregulation by active MMP9.

Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

Ca2+ binding to the ubiquitous Ca2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca2+-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K+ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K+ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca2+-dependent gating process.

Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling.

There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein-protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells, and co-immunoprecipitation using StrepTag-labeled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase, the first enzyme of the DNPB pathway, we discovered PAICS interacts with all other known DNPB enzymes and with MTHFD1, an enzyme which supplies the 10-formyltetrahydrofolate cofactor essential for DNPB. We show these interactions are present in cells grown in both purine-depleted and purine-rich conditions, suggesting at least a partial assembly of these enzymes may be present regardless of the activity of the DNPB pathway. We also demonstrate that tagging of PAICS on its C terminus disrupts these interactions and that this disruption is correlated with disturbed DNPB activity. Finally, we show that crPAICS cells with reintegrated N-terminally tagged PAICS regained effective DNPB with metabolic signatures of channeled synthesis, whereas crPAICS cells that reintegrated C-terminally tagged PAICS exhibit reduced DNPB intermediate pools and a perturbed partitioning of inosine monophosphate into AMP and GMP. Our results provide molecular evidence in support of purinosomes and suggest perturbing PPIs between DNPB enzymes negatively impact metabolite flux through this important pathway.

Liguleless narrow and narrow odd dwarf act in overlapping pathways to regulate maize development and metabolism.

Narrow odd dwarf (nod) and Liguleless narrow (Lgn) are pleiotropic maize mutants that both encode plasma membrane proteins, cause similar developmental patterning defects, and constitutively induce stress signaling pathways. To investigate how these mutants coordinate maize development and physiology, we screened for protein interactors of NOD by affinity purification. LGN was identified by this screen as a strong candidate interactor, and we confirmed the NOD-LGN molecular interaction through orthogonal experiments. We further demonstrated that LGN, a receptor-like kinase, can phosphorylate NOD in vitro, hinting that they could act in intersecting signal transduction pathways. To test this hypothesis, we generated Lgn-R;nod mutants in two backgrounds (B73 and A619), and found that these mutations enhance each other, causing more severe developmental defects than either single mutation on its own, with phenotypes including very narrow leaves, increased tillering, and failure of the main shoot. Transcriptomic and metabolomic analyses of the single and double mutants in the two genetic backgrounds revealed widespread induction of pathogen defense genes and a shift in resource allocation away from primary metabolism in favor of specialized metabolism. These effects were similar in each single mutant and heightened in the double mutant, leading us to conclude that NOD and LGN act cumulatively in overlapping signaling pathways to coordinate growth-defense tradeoffs in maize.

Chromatin regulator Eaf3p regulates nitrogen metabolism in Saccharomyces cerevisiae as a trans-acting factor.

IMPORTANCE: In this study, the mechanism of chromatin regulator Eaf3p regulating nitrogen metabolism in S. cerevisiae was investigated. It provides theoretical support for epigenetic modifications of cells to alter the level of histone modifications, coordinate the expression of multiple genes, and make it more conducive to the co-metabolism of multiple nitrogen sources. Moreover, it provides new ideas for industrial brewing yeast strains to achieve nitrogen source metabolism balance, reduce the accumulation of harmful nitrogen metabolites, and improve fermentation efficiency. This study provides a reference for changing the performance of microbial strains and improving the quality of traditional fermentation products and provides a theoretical basis for studying epigenetic modification and nitrogen metabolism regulation. It has an important theoretical explanation and practical application value. In addition, this study also provides useful clues for the study.

Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres: Design, Optimization and Integration.

This work demonstrates the potential of calcium- and nickel-crosslinked Gellan Gum (GG) microspheres to capture the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) directly from complex Komagataella pastoris mini-bioreactor lysates in a batch method. Calcium-crosslinked microspheres were applied in an ionic exchange strategy, by manipulation of pH and ionic strength, whereas nickel-crosslinked microspheres were applied in an affinity strategy, mirroring a standard immobilized metal affinity chromatography. Both formulations presented small diameters, with appreciable crosslinker content, but calcium-crosslinked microspheres were far smoother. The most promising results were obtained for the ionic strategy, wherein calcium-crosslinked GG microspheres were able to completely bind 0.1% (v/v) DM solubilized STEAP1 in lysate samples (~7 mg/mL). The target protein was eluted in a complexed state at pH 11 with 500 mM NaCl in 10 mM Tris buffer, in a single step with minimal losses. Coupling the batch clarified sample with a co-immunoprecipitation polishing step yields a sample of monomeric STEAP1 with a high degree of purity. For the first time, we demonstrate the potential of a gellan batch method to function as a clarification and primary capture method towards STEAP1, a membrane protein, simplifying and reducing the costs of standard purification workflows.

TBK1 interacts with tau and enhances neurodegeneration in tauopathy.

One of the defining pathological features of Alzheimer's disease (AD) is the deposition of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau in the brain. Aberrant activation of kinases in AD has been suggested to enhance phosphorylation and toxicity of tau, making the responsible tau kinases attractive therapeutic targets. The full complement of tau-interacting kinases in AD brain and their activity in disease remains incompletely defined. Here, immunoaffinity enrichment coupled with mass spectrometry (MS) identified TANK-binding kinase 1 (TBK1) as a tau-interacting partner in human AD cortical brain tissues. We validated this interaction in human AD, familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) caused by mutations in MAPT (R406W & P301L) and corticobasal degeneration (CBD) postmortem brain tissues as well as human cell lines. Further, we document increased TBK1 activation in both AD and FTDP-17 and map TBK1 phosphorylation sites on tau based on in vitro kinase assays coupled to MS. Lastly, in a Drosophila tauopathy model, activating expression of a conserved TBK1 ortholog triggers tau hyperphosphorylation and enhanced neurodegeneration, whereas knockdown had the reciprocal effect, suppressing tau toxicity. Collectively, our findings suggest that increased TBK1 activation may promote tau hyperphosphorylation and neuronal loss in AD and related tauopathies.

PRPF31 interacts with PRPH2 confirmed by co-immunoprecipitation and co-localization.

Both PRPF31 and PRPH2 are the causative genes for retinitis pigmentosa. And both of them are associated with the balance of rhodopsin. In this study, we aim to investigate the co-expression and interaction of PRPF31 and PRPH2. We used PRPF31-eGFP, PRPF31-3xFlag and PRPH2-mCherry vectors were transfected into HEK293T and APRE-19 cells. Immunoblotting and co-immunoprecipitation (Co-IP) were used for gene expression validation and protein interaction. Immunofluorescence staining assay was used to test the co-localization analysis of PRPF31 and PRPH2. Co-IP experiments showed that PRPF31 could be pulled down with an anti-PRPH2 antibody. There was co-localization between PRPF31 and PRPH2 in HEK293T, APRE-19 and mouse retina. The Co-IP and co-localization experiments suggest that PRPF31 interacted with PRPH2.

VvTOR interacts with VvSnRK1.1 and regulates sugar metabolism in grape.

MAIN CONCLUSION: VvTOR interacts with VvSnRK1.1 and regulates sugar accumulation and sugar-related genes expression in grape. Target of rapamycin (TOR) and sucrose-non-fermenting-related protein kinase 1.1 (SnRK1.1) both are critical proteins in plant sugar metabolism. Glucose-TOR signaling dictates transcriptional reprogramming of gene sets involved in central and secondary metabolism, cell cycle, transcription, signaling, transport and folding. SnRK1.1 is involved in sucrose-induced hypocotyl elongation. However, the relationship of TOR and SnRK1.1 in regulating sugar metabolism is unclear. In the study, we utilized grape (Vitis vinifera) calli to explore the relationship between TOR and SnRK1.1 in the sugar metabolism. We found that VvTOR interacted with VvSnRK1.1. By subcellular localization, VvTOR was found in the nucleus and cell membrane. Transgenic grape calli achieved by Agrobacterium-mediated transformation contained less glucose compared to WT calli. The fructose contents were markedly increased in the overexpressing VvTOR (OE-VvTOR), OE-VvTOR + RNAi-VvSnRK1.1 and RNAi-VvTOR + OE-VvSnRK1.1 transgenic calli. Sucrose contents were significantly increased in the OE-VvTOR transgenic calli and reduced in the OE-VvTOR + RNAi-VvSnRK1.1 transgenic calli, which implied that the pathway of VvTOR improving sucrose content might need the expression of VvSnRK1.1. VvTOR interacted with VvSnRK1.1 and regulated sugar metabolism in grape. These results suggest that there is a crosstalk between TOR and SnRK1.1 in plant sugar metabolism.

Development and Validation of Arc Nanobodies: New Tools for Probing Arc Dynamics and Function.

Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions.

Schistosoma japonicum proteins that interact with the gynecophoral canal protein identified using a yeast two-hybrid system.

The large amount of schistosome eggs produced by mature female worms not only induce major pathological damage to the host but also lead to the transmission of schistosomiasis. Mature female schistosome worms need constant pairing contact with a male partner as male signaling is indispensable to female growth, development, and reproduction. The gynecophoral canal protein (GCP), a cell-surface glycoprotein, plays a potential role in the interaction between males and females and in stimulating female development and maturation. In this study, a yeast two-hybrid cDNA library of Schistosoma japonicum (Sj) parasites 18 days post-infection (dpi) was constructed; the Sjgcp gene was inserted into a pGBKT7-BD bait plasmid and used as a bait protein to screen for its molecular interactions using a yeast mating procedure. Twenty-four prey proteins that interacted with the SjGCP were selected after excluding false positives; the interactions between S.japonicum lethal giant larvae (SjLGL) and SjGCP, S.japonicum type V collagen (SjColV) and SjGCP, were verified by co-immunoprecipitation. The RNA interference against SjGCP, SjColV and SjGCP + SjColV led to severe underdevelopment of tegument in male worms and vitelline globules in female worms as well as reduced reproductive capacity of the females. Collectively, SjGCP and its interacting proteins may play pivotal roles in growth and development. The findings also suggested that SjGCP and its interacting protein partners might represent new candidate targets for drug development against schistosomiasis.

Dysregulation of Oxygen Sensing/Response Pathways in Pregnancies Complicated by Idiopathic Intrauterine Growth Restriction and Early-Onset Preeclampsia.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are the leading causes of maternal and fetal morbidity/mortality. The central deficit in both conditions is impaired placentation due to poor trophoblast invasion, resulting in a hypoxic milieu in which oxidative stress contributes to the pathology. We examine the factors driving the hypoxic response in severely preterm PE (n = 19) and IUGR (n = 16) placentae compared to the spontaneous preterm (SPT) controls (n = 13) using immunoblotting, RT-PCR, immunohistochemistry, proximity ligation assays, and Co-IP. Both hypoxia-inducible factor (HIF)-1α and HIF-2α are increased at the protein level and functional in pathological placentae, as target genes prolyl hydroxylase domain (PHD)2, PHD3, and soluble fms-like tyrosine kinase-1 (sFlt-1) are increased. Accumulation of HIF-α-subunits occurs in the presence of accessory molecules required for their degradation (PHD1, PHD2, and PHD3 and the E3 ligase von Hippel-Lindau (VHL)), which were equally expressed or elevated in the placental lysates of PE and IUGR. However, complex formation between VHL and HIF-α-subunits is defective. This is associated with enhanced VHL/DJ1 complex formation in both PE and IUGR. In conclusion, we establish a significant mechanism driving the maladaptive responses to hypoxia in the placentae from severe PE and IUGR, which is central to the pathogenesis of both diseases.

A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34+-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis.

Regulating Effect of Cytochrome b5 Overexpression on Human Breast Cancer Cells.

Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b5 (Cyt b5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b5 content. Based on the low content of Cyt b5 in MDA-MB-231 cells, the overexpression of Cyt b5 was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b5 MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b5 most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b5 has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b5 and provides clues for clinical treatment.

Andrographolide Induces G2/M Cell Cycle Arrest and Apoptosis in Human Glioblastoma DBTRG-05MG Cell Line via ERK1/2 /c-Myc/p53 Signaling Pathway.

Human glioblastoma multiforme (GBM) is one of the most malignant brain tumors, with a high mortality rate worldwide. Conventional GBM treatment is now challenged by the presence of the blood-brain barrier (BBB), drug resistance, and post-treatment adverse effects. Hence, developing bioactive compounds isolated from plant species and identifying molecular pathways in facilitating effective treatment has become crucial in GBM. Based on pharmacodynamic studies, andrographolide has sparked the interest of cancer researchers, who believe it may alleviate difficulties in GBM therapy; however, it still requires further study. Andrographolide is a bicyclic diterpene lactone derived from Andrographis paniculata (Burm.f.) Wallich ex Nees that has anticancer properties in various cancer cell lines. The present study aimed to evaluate andrographolide's anticancer effectiveness and potential molecular pathways using a DBTRG-05MG cell line. The antiproliferative activity of andrographolide was determined using the WST-1 assay, while scratch assay and clonogenic assay were used to evaluate andrographolide's effectiveness against the cancer cell line by examining cell migration and colony formation. Flowcytometry was also used to examine the apoptosis and cell cycle arrest induced by andrographolide. The mRNA and protein expression level involved in the ERK1/2/c-Myc/p53 signaling pathway was then assessed using qRT-PCR and Western blot. The protein-protein interaction between c-Myc and p53 was determined by a reciprocal experiment of the co-immunoprecipitation (co-IP) using DBTRG-05MG total cell lysate. Andrographolide significantly reduced the viability of DBTRG-05MG cell lines in a concentration- and time-dependent manner. In addition, scratch and clonogenic assays confirmed the effectiveness of andrographolide in reducing cell migration and colony formation of DBTRG-05MG, respectively. Andrographolide also promoted cell cycle arrest in the G2/M phase, followed by apoptosis in the DBTRG-05MG cell line, by inducing ERK1/2, c-Myc, and p53 expression at the mRNA level. Western blot results demonstrated that c-Myc overexpression also increased the production of the anti-apoptotic protein p53. Our findings revealed that c-Myc and p53 positively interact in triggering the apoptotic signaling pathway. This study successfully discovered the involvement of ERK1/2/c-Myc/p53 in the suppression of the DBTRG-05MG cell line via cell cycle arrest followed by the apoptosis signaling pathway following andrographolide treatment.