Target Proteins In Cells Research Articles

Glucose Transporter-Targeting Chimeras Enabling Tumor-Selective Degradation of Secreted and Membrane Proteins.

Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.

Gene Editing by Ferrying of CRISPR/Cas Ribonucleoprotein Complexes in Enveloped Virus-Derived Particles.

The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for invivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted invivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.

Exploration of small molecule compounds targeting abdominal aortic aneurysm based on CMap database and molecular dynamics simulation.

The mitigation of abdominal aortic aneurysm (AAA) growth through pharmaceutical intervention offers the potential to avert the perils associated with AAA rupture and the subsequent need for surgical intervention. Nevertheless, the existing effective drugs for AAA treatment are limited, necessitating a pressing exploration for novel therapeutic medications. AAA-related transcriptome data were downloaded from GEO, and differentially expressed genes (DEGs) in AAA tissue were screened for GO and KEGG enrichment analyses. Small molecule compounds and their target proteins with negative connectivity to the AAA expression profile were predicted in the Connectivity Map (CMap) database. Molecular docking and molecular dynamics simulation were performed to predict the binding of the target protein to the small molecule compound, and the MM/GBSA method was used to calculate the binding free energy. Cluster analysis was performed using the cluster tool in the GROMACS package. An AAA cell-free model was built, and CETSA experiments were used to demonstrate the binding ability of small molecules to the target protein in cells. A total of 2244 DEGs in AAA were obtained through differential analysis, and the DEGs were mainly enriched in the tubulin binding biological function and cell cycle pathway. The CMap results showed that Apicidin had a potential therapeutic effect on AAA with a connectivity score of -97.74, and HDAC4 was the target protein of Apicidin. Based on literature, HDAC4-Apicidin was selected as the subsequent research object. The lowest affinity of Apicidin-HDAC4 molecular docking was -8.218kcal/mol. Molecular dynamics simulation results indicated that Apicidin-HDAC4 could form a stable complex. MM/GBSA analysis showed a total binding free energy of -55.40 ± 0.79kcal/mol, and cluster analysis showed that there were two main conformational clusters during the binding process, accounting for 22.4% and 57.8%, respectively. Apicidin could form hydrogen bonds with surrounding residues for stable binding. CETSA experiment proved the stable binding ability of Apicidin and HDAC4. Apicidin inhibited HDAC4 in AAA and exhibited favorable protein-ligand interactions and stability, making it a potential candidate drug for treating AAA.

A comprehensive view on the fisetin impact on colorectal cancer in animal models: Focusing on cellular and molecular mechanisms.

Flavonoids, including fisetin, have been linked to a reduced risk of colorectal cancer (CRC) and have potential therapeutic applications for the condition. Fisetin, a natural flavonoid found in various fruits and vegetables, has shown promise in managing CRC due to its diverse biological activities. It has been found to influence key cell signaling pathways related to inflammation, angiogenesis, apoptosis, and transcription factors. The results of this study demonstrate that fisetin induces colon cancer cell apoptosis through multiple mechanisms. It impacts the p53 pathway, leading to increased levels of p53 and decreased levels of murine double minute 2, contributing to apoptosis induction. Fisetin also triggers the release of important components in the apoptotic process, such as second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI and cytochrome c. Furthermore, fisetin inhibits the cyclooxygenase-2 and wingless-related integration site (Wnt)/epidermal growth factor receptor/nuclear factor kappa B signaling pathways, reducing Wnt target gene expression and hindering colony formation. It achieves this by regulating the activities of cyclin-dependent kinase 2 and cyclin-dependent kinase 4, reducing retinoblastoma protein phosphorylation, decreasing cyclin E levels, and increasing p21 levels, ultimately influencing E2 promoter binding factor 1 and cell division cycle 2 (CDC2) protein levels. Additionally, fisetin exhibits various effects on CRC cells, including inhibiting the phosphorylation of Y-box binding protein 1 and ribosomal S6 kinase, promoting the phosphorylation of extracellular signal-regulated kinase 1/2, and disrupting the repair process of DNA double-strand breaks. Moreover, fisetin serves as an adjunct therapy for the prevention and treatment of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA)-mutant CRC, resulting in a reduction in phosphatidylinositol-3 kinase (PI3K) expression, Ak strain transforming phosphorylation, mTOR activity, and downstream target proteins in CRC cells with a PIK3CA mutation. These findings highlight the multifaceted potential of fisetin in managing CRC and position it as a promising candidate for future therapy development.

The Binding Capacity of the Lead Phytochemical Molecule to Cancer Cell Target Proteins and its Potential Anticancer Properties with Respect to Standard Drugs

Molecular docking is an important tool for connecting molecular biological structure to computer-aided drug design. The purpose of this blind docking experiment is to compare the binding energies of these three drugs and to predict the most likely binding poses of a ligand with a known three dimensional structure of a protein. To substantiate our previous in vitro study findings, an in silico model was chosen to compare the binding properties of the three drugs. The work is entirely bioinformatics in nature. Blind docking was accomplished with the help of free software/(s). A comparison was made among the three drgus, used primarily in cancer treatment, namely, anastrazole, capecitabine and quercetin. In our in vitro study, these three drugs were extremely effective. CB dock-2 to blind dock, SwissTargetPrediction to choose the targets, PubChem and Protein Data Base to obtain 3D structures of ligand and target respectively were used. Absorption, Distribution, Metabolism, and Excretion (ADME) from SwissADME, drug likeness from MolSoft L.L.C. software, and Liponski’s rule was used to check the “Rule of five (RO5)”. The crystallographic structures have resolution values ranging from 1 Å to 3 Å. Lipinski’s rule of five, Swiss ADME and drug likeness were used to compare the three drugs. For all of the targets studied, quecetin appeared to have the highest AutoDock vina scores. To summarize the current findings and publicly available data, quercetin is chemoprotective and radioprotective to healthy/ normal cells and it can be used during cancer treatment.

Aptamer and phosphorothioated DNA engineered liposomes as a targeted intracellular protein delivery system

Targeted delivery of proteins into desired cell groups is crucial for disease therapy and cellular functionalization. However, current delivery methods face severe side effects and off-target problems, making it difficult to achieve cell-targeted protein delivery. Herein, we developed a user-friendly membrane fusion liposome for cancer cell-targeted protein delivery. Phosphorothioated DNA-mediated membrane fusion was employed as an efficient transmembrane delivery approach. The phosphorothioated DNA was capped with the AS1411 aptamer, which specifically recognizes nucleolar proteins on the cancer cell membrane, enabling a controllable delivery of proteins into targeted cancer cells. This delivery system exhibited commendable biocompatibility and targeted delivery ability, thereby realizing highly effective cancer cell inhibition in vitro. The in vivo results further suggested that the membrane protein-responsive membrane fusion delivery system offers a new avenue for highly biocompatible and targeted protein delivery.

The NeuroProtect Formula: A Preventive Approach to AD Targeting the HIF-1/PI3K-AKT Signaling Pathway Evaluated through In Vivo, In Vitro, and Network Pharmacology Approaches.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder with limited options for reversing its middle-to-late stages. Early intervention is crucial to slow down disease progression. This study aimed to investigate the potential of the NeuroProtect (NP) formula, a combination of geniposide and Panax notoginseng saponins, in preventing AD. We evaluated the effects of the NP formula on amyloid plaque accumulation, neuronal degeneration, and molecular signaling pathways using in vivo and in vitro models. To predict functional pathways and potential downstream targets of NP intervention, we employed network pharmacology. The preventative impact of the NP formula was assessed using APP/PS1 mice. We conducted HE staining, ELISA assay, Golgi staining, and immunohistochemistry to detect the protective effect of NP. Additionally, cell experiments were performed to assess cell activity and target protein expression. Network pharmacology analysis revealed 145 drug-disease interactions and identified 5 core active targets associated with AD. Molecular docking results demonstrated strong binding affinity between the components of the NP formula (GP, GN-Rb1, GN-Rg1, NS-R1) and target proteins (STAT3, HIF1A, TLR4, mTOR, VEGFA). Notably, the binding energy between NS-R1 and mTOR was -11.4kcal/mol. Among the top 10 enriched KEGG pathways, the HIF-1 and PI3K-AKT signaling pathways were highlighted. In vivo experiments demonstrated that the NP formula significantly ameliorated pathological changes, decreased the Aβ42/Aβ40 ratio in the hippocampus and cortex, and increased dendritic spine density in the CA1 region during the early stage of AD. In vitro experiments further illustrated the NP formula's ability to reverse the inhibitory effects of Aβ25-35 on cell viability and regulate the expression of Tlr4, Mtor, Hif1a, Stat3, and Vegfa. Our findings suggest that NP exhibits neuroprotective effects during the early stages of AD, positioning it as a potential candidate for AD prevention. The NP formula may exert its preventive effects through the HIF-1/PI3K-AKT signaling pathway, with mTOR identified as a key target.

Selective Elimination of Senescent Cancer Cells by Galacto-Modified PROTACs.

Although the selective and effective clearance of senescent cancer cells can improve cancer treatment, their development is confronted by many challenges. As part of efforts designed to overcome these problems, prodrugs, whose design is based on senescence-associated β-galactosidase (SA-β-gal), have been developed to selectively eliminate senescent cells. However, chemotherapies relying on targeted molecular inhibitors as senolytic drugs can induce drug resistance. In the current investigation, we devised a new strategy for selective degradation of target proteins in senescent cancer cells that utilizes a prodrug composed of the SA-β-gal substrate galactose (galacto) and the proteolysis-targeting chimeras (PROTACs) as senolytic agents. Prodrugs Gal-ARV-771 and Gal-MS99 were found to display senolytic indexes higher than those of ARV-771 and MS99. Significantly, results of in vivo studies utilizing a human lung A549 xenograft mouse model demonstrated that concomitant treatment with etoposide and Gal-ARV-771 leads to a significant inhibition of tumor growth without eliciting significant toxicity.

Secondary reagents for immunohistochemical research of rat brain

BACKGROUND: The critical factor when working with immunohistochemistry in laboratory animals is to select appropriate secondary antibodies, that allow clear and specific visualization of tissue antigens. Many reliable secondary reagents are currently not available for purchase, which determines the high relevance of replacing them with other detection systems.AIM: To verify the effectiveness of available secondary reagents for immunohistochemical research of the rat brain.METHODS: Brain samples from Wistar and SHR rats (n=4) were used for the study. Iba-1, GFAP and vimentin immunohistochemistry was carried out using various polymer-based detection systems, namely UltraVision Quanto, N-Histofine Simple stain MAX PO ® and UnoVue Rabbit HRP.RESULTS: All three studied polymer systems demonstrated visualisation of target proteins in brain tissues and cells corresponding to the general understanding of structures containing Iba-1, GFAP and vimentin. The UnoVue Rabbit HRP and Quanto Detection System HRP kits showed good and similarly specific immunohistochemical reaction. However, the UnoVue Rabbit HRP kit was less sensitive compared to the Quanto Detection System HRP. The reaction with N-Histofine® Simple Stain MAX PO was not optimal due to the presence of non-specific background staining that was not present with other reagents.CONCLUSION: Two secondary antibody kits from the three studied showed optimal efficiency of immunohistochemical reaction and minimal background staining. N-Histofine® Simple Stain MAX PO is not suitable for immunohistochemical research of rat brain tissue.

Construction of an Escherichia coli chassis for efficient biosynthesis of human-like N-linked glycoproteins.

The production of N-linked glycoproteins in genetically engineered Escherichia coli holds significant potential for reducing costs, streamlining bioprocesses, and enhancing customization. However, the construction of a stable and low-cost microbial cell factory for the efficient production of humanized N-glycosylated recombinant proteins remains a formidable challenge. In this study, we developed a glyco-engineered E. coli chassis to produce N-glycosylated proteins with the human-like glycan Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, containing the human glycoform Gal-β-1,4-GlcNAc-β-1,3-. Our initial efforts were to replace various loci in the genome of the E. coli XL1-Blue strain with oligosaccharyltransferase PglB and the glycosyltransferases LsgCDEF to construct the E. coli chassis. In addition, we systematically optimized the promoter regions in the genome to regulate transcription levels. Subsequently, utilizing a plasmid carrying the target protein, we have successfully obtained N-glycosylated proteins with 100% tetrasaccharide modification at a yield of approximately 320mg/L. Furthermore, we constructed the metabolic pathway for sialylation using a plasmid containing a dual-expression cassette of the target protein and CMP-sialic acid synthesis in the tetrasaccharide chassis cell, resulting in a 40% efficiency of terminal α-2,3- sialylation and a production of 65mg/L of homogeneously sialylated glycoproteins in flasks. Our findings pave the way for further exploration of producing different linkages (α-2,3/α-2,6/α-2,8) of sialylated human-like N-glycoproteins in the periplasm of the plug-and-play E. coli chassis, laying a strong foundation for industrial-scale production.

Multiplex Cyclic Fluorescent Immunohistochemistry.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

A protein-miRNA biomic analysis approach to explore neuroprotective potential of nobiletin in human neural progenitor cells (hNPCs).

Chemical-induced neurotoxicity is increasingly recognized to accelerate the development of neurodegenerative disorders (NDs), which pose an increasing health burden to society. Attempts are being made to develop drugs that can cross the blood-brain barrier and have minimal or no side effects. Nobiletin (NOB), a polymethoxylated flavonoid with anti-oxidative and anti-inflammatory effects, has been demonstrated to be a promising compound to treat a variety of NDs. Here, we investigated the potential role of NOB in sodium arsenate (NA)-induced deregulated miRNAs and target proteins in human neural progenitor cells (hNPCs). The proteomics and microRNA (miRNA) profiling was done for different groups, namely, unexposed control, NA-exposed, NA + NOB, and NOB groups. Following the correlation analysis between deregulated miRNAs and target proteins, RT-PCR analysis was used to validate the selected genes. The proteomic analysis showed that significantly deregulated proteins were associated with neurodegeneration pathways, response to oxidative stress, RNA processing, DNA repair, and apoptotic process following exposure to NA. The OpenArray analysis confirmed that NA exposure significantly altered miRNAs that regulate P53 signaling, Wnt signaling, cell death, and cell cycle pathways. The RT-PCR validation studies concur with proteomic data as marker genes associated with autophagy and apoptosis (HO-1, SQSTM1, LC-3, Cas3, Apaf1, HSP70, and SNCA1) were altered following NA exposure. It was observed that the treatment of NOB significantly restored the deregulated miRNAs and proteins to their basal levels. Hence, it may be considered one of its neuroprotective mechanisms. Together, the findings are promising to demonstrate the potential applicability of NOB as a neuroprotectant against chemical-induced neurotoxicity.

A comprehensive review on lncRNA LOXL1-AS1: molecular mechanistic pathways of lncRNA LOXL1-AS1 in tumorigenicity of cancer cells.

Long non-coding RNAs (lncRNAs) are versatile RNAs that regulate various cellular processes, such as gene regulation, by acting as signals, decoys, guides, and scaffolds. A novel recognized lncRNA, LOXL1-antisense RNA 1 (LOXL1-AS1), is dysregulated in some diseases, including cancer, and acts as an oncogenic lncRNA in many types of cancer cells. Upregulation of LOXL1-AS1 has been involved in proliferation, migration, metastasis, and EMT, as well as inhibiting apoptosis in cancer cells. Most importantly, the malignant promoting activity of LOXL1-AS1 can be mostly mediated by sequestering specific miRNAs and inhibiting their binding to the 3´UTR of their target mRNAs, thereby indirectly regulating gene expression. Additionally, LOXL1-AS1 can decoy transcription factors and proteins and prevent their binding to their regulatory regions, inhibiting their mechanistic activity on the regulation of gene expression and signaling pathways. This review presents the mechanistic pathways of the oncogenic role of LOXL1-AS1 by modulating its target miRNAs and proteins in various cancer cells. Having information about the molecular mechanisms regulated by LOXL1-AS1 in cancer cells can open ways to find out particular prognostic biomarkers, as well as discover novel therapeutic approaches for different types of cancer.

Selective inhibition of cancer cell migration using a pH-responsive nucleobase-modified DNA aptamer.

Because of the extracellular acidic microenvironment of cancer cells, many pH-responsive molecules have become indispensable materials for bioanalysis and targeted therapy development. pH-Responsive DNA aptamers, which selectively bind to target proteins in cancer cells, have become a key research target in the therapeutic field. However, conventional pH-responsive aptamers have fatal drawbacks, such as complex structures, sequence limitation, and difficulties in mass production, as they require special nucleic acid structures, including the i-motif and DNA triplex. To address these issues, we utilized AnC, which is an unnatural nucleobase with a pK aH of 5.9, to construct a simple pH-responsive DNA aptamer (CSL1-II) for selective binding to the c-Met protein expressed in cancer cells. CSL1-II in a weakly acidic environment had a stronger inhibitory effect on the HGF/c-Met pathway and exerted a strong controlling effect on the spreading and migration of cancer cells. Our strategy provides a simple and versatile method to develop pH-responsive DNA aptamers and represents the first example of a cancer-selective c-Met antagonist that inhibits cell migration.

Antimicrobial Activity and Molecular Docking of Benzoyl-N,N’-dialkylurea against Target Proteins in Microbial Cells

This research aims to evaluate the antimicrobial activity of benzoyl dialkylurea derivatives to meet the global need for new antibiotic lead compounds. Four compounds were tested in vitro against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans by broth dilution method using ciprofloxacin and nystatin as standards. Docking simulations were performed to target essential proteins including DNA gyrase, FabH, RNA polymerase in bacterial cells and DHFR in C. albicans. BDMU, BEU1, BEU2, BEU3 exhibited antibacterial activity while BEU1 and BEU2 showed weak antifungal activity against C. albicans. The BDMU, BEU2, BEU3 considered promising growth inhibition against P. aeruginosa. In silico molecular docking on DNA gyrase from P. aeruginosa (PDB. 6M1S) was proposed as a model for mechanism of action in bacterial cells. The monobenzoyl of dialkylurea containing urea functionality with chloro-substituent at position-2 and 4 on aromatic rings were potential as lead compound to generate new antibacterial agent.

Targeting AR-positive breast cancer cells via drug repurposing approach

Androgen Receptor (AR) is overexpressed in almost all the molecular subtypes of breast cancer. Besides aiding the tumorigenic environment of cancer by abnormal cell proliferation, AR also takes part in promoting cancer signaling pathways, thereby promoting aggressiveness. In this study, AR was selected as the target protein in breast cancer cells. Following this, a library of 1293 FDA-approved drugs was screened via molecular docking, MD simulation, and MMPBSA binding energy. Amongst the library of compounds, Adapalene exhibited the least binding energy of (−10.2 kCal/mol) in comparison to that of the chosen reference compound, Nilutamide (−8.6 kCal/mol). Furthermore, the in vitro efficacy of Adapalene was also determined in two different breast cancer cell lines such as MCF7 (AR-positive/ER-positive) and MDA-MB-231 (AR negative/TNBC). Initially, the cell viability assay (MTT) was performed, which endowed us with a lesser IC50 value of Adapalene in comparison to Nilutamide in both cell lines. The IC50 of Adapalene was found to be 12 μM and 39.4 μM in MCF7 and MDA-MB-231 cells, respectively. Furthermore, Adapalene also induced cellular ROS and apoptosis by 3.5-fold and 26.58% in MCF7 cells. However, the overall effect of Adapalene was significantly lower in the case of MDA-MB-231 cell lines, which could be attributed to its inherent nature of the absence of hormone receptors. Conclusively, Adapalene possesses greater therapeutic efficacy in comparison to the control drug, thereby hinting towards the potential use of Adapalene in the treatment of AR-positive breast cancer.

Role of a cellular thermal shift assay on evaluation of drug‐target engagement for compound selection

AbstractBackgroundIdentification of small molecule‐target engagement is important for target‐based drug discovery. To support drug discovery efforts for Alzheimer’s Disease, we developed and implemented cellular thermal shift assays (CETSA) based on HiBiT/LgBiT reconstituted NanoLuc method (Promega). Advantages of the CETSA are testing target engagement in an intact cellular context, simple mix‐and‐read procedures, and high capacity to meet drug discovery project needs.MethodStable cell lines (HEK293t or HMC3) expressing HiBit (11 amino acids; Promega) tagged target were established. The CETSA uses Nano‐Glo HiBiT Lytic Detection kit (Promega) to detect abundance of undenatured HiBit‐tagged target protein in cells, while heat caused protein aggregates to be mostly undetectable. The CETSA procedures are 1) plating cells into 96‐well PCR plates; 2) treating cells with compounds for 60 min in 37 oC incubator; 3) heating cell plates for 3 min in a PCR machine; 4) processing with Nano‐Glo HiBiT Lytic Detection kit (10 min) and reading plates for luminescence. Before testing compounds, an Tagg (temperature at 50% maximal signal during aggregation) for each target was determined and used as baseline. When a compound engages the target, it caused Tagg shifts that can be quantified by the assay.Result1) Tagg is target specific, with INPP5D Tagg = 44.2 °C and PLCG2 Tagg = 45.9 °C. 2) Screening compounds at 100 µM on cells and heated at Tagg, the luminescent signal changes (compared to untreated control) suggesting compound‐target engagement. 3) The CETSA with compound dosing and heating at Tagg was used to confirm the compound engagement. 4) The HiBit tag control CETSA with compound dosing and Tagg heating was used to find non‐specific binders. The assay platform has been implemented for drug discovery efforts targeting INPP5D and PLCG2 respectively; example data will be shown.ConclusionThe CETSA platform is efficient, reproducible and has high capacity, making it well‐suited to target based drug discovery research. Application of the CETSA on projects targeting INPP5D, PLCG2 of microglia has facilitated compound selection based on confirming molecule target engagement, which allowed our project team to quickly focus on selected compounds for further studies.

The subset of peroxisomal tail-anchored proteins do not reach peroxisomes via ER, instead mitochondria can be involved.

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.

An unbiased approach of individual tissue-specific autoantibody identification using Immunoprecipitation coupled to Mass-Spectrometry (IP-MS)

Abstract Background and aim Autoreactive antibodies have gained increasing recognition in autoimmune diseases of the central nervous system (CNS) serving as disease drivers and / or biomarkers. Identification of novel autoantibodies therefore is crucial to optimize diagnostic work and therapy of these diseases as well as understanding their pathogenesis. Hence, we aimed to develop an unbiased strategy to identify patients’ individual main autoantibodies directed against brain epitopes. Methods We used murine tissue-based assays to screen patients’ sera and cerebrospinal fluids (CSFs) for autoreactive IgGs with known and unknown antibody targets. IgGs from autoreactive patients’ samples were coupled to magnetic beads and used as a bait to pull down potential targets from homogenized brain tissue of wildtype mice. After precipitation and several washes to reduce unspecific binding, beads were subjected to on-bead tryptic digestion followed by mass spectrometry based proteomic analyses using liquid chromatography tandem mass-spectrometry (LC-MS/MS). For antibody targets identification the fold change enrichment compared to a negative control or to all other pulldown samples, as well as the protein abundance was used. Antibody target validation was done via recombinant expression of the target protein in HEK-293 cells and neutralization with the respective antigen in tissue-based immunofluorescence assays. Results As proof of principle, we were able to identify Grin1 Caspr2, Trim46 and Nf-h [MOU1] as main targets of patients’ sera or CSF with autoimmune encephalopathy. All four antigens showed highest enrichment and/or highest protein abundance in the respective samples and have been confirmed in cell-based assays. In addition, we identified one novel anti-neuronal antibody target in serum and CSF of a patient showing a speckled staining of the granular cerebellar layer. Preabsorbance of the serum and CSF with the recombinant protein fully neutralized the signal of the tissue-based immunofluorescence. Conclusions Our IP-MS approach is suitable to identify the main autoantibody targets in patients’ serum and CSF within murine CNS tissue thereby providing an unbiased and scalable technique allowing for tissue-specific autoantibody determination.

Loss of DDX3X Function Promotes CLL Progression By Facilitating NOTCH1 mRNA Translation

A variety of driver genes are present in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and those with high mutation frequencies include SF3B1, NOTCH1, and TP53, which are involved in the development of CLL/SLL and are associated with poor prognosis. The biological significance of some driver genes, such as DDX3X, has not yet been elucidated. DDX3X is located on the X chromosome. As one of the members of the DEAD-box helicase family, it is expressed ubiquitously in human tissues and participates in many biological processes. Here, we described the mapping of the DDX3X abnormalities and demonstrated its clinical significance in CLL/SLL. We also explored the effect and mechanism of DDX3X dysregulation on tumorigenesis and development of CLL by in vitro experiments. To examine the mutational landscape of DDX3X, we performed next-generation sequencing on 402 CLL/SLL patients, and 17 patients (4.2%) had DDX3X mutations. Six patients were frameshift mutations and 2 were nonsense mutation, which caused truncation of DDX3X. Among the 7 cases of missense mutations, 4 cases involved the ATP-binding or C-terminal domain, which may affect the function of DDX3X. The remaining two cases are deletion mutation and splicing mutation. Next, we evaluated the clinical significance and prognostic role of DDX3X mutation. The patients with DDX3X mutations are mostly male (94.1% vs 66.5%, P=0.017), and had more adverse prognostic indicators: unmutated IGHV (88.2% vs 48.6%, P=0.002), 17p deletion (42.9% vs 11.4%, P=0.041), and 11q deletion (46.7% vs 15.7%, P=0.006). DDX3X mutation was an independent risk factor for disease specific survival of CLL/SLL in the competing-risk regression. Also, we found all (14/14) the mutated patients and 15.2% (5/33) of the non-mutated patients had low expression of DDX3X in bone marrow samples by immunohistochemistry. Overall, these results indicates that dysregulation of DDX3X are common in CLL/SLL and may contribute to its progression. To delineate the functional consequences of DDX3X loss in CLL, we used CRISPR/cas9 system to construct stable DDX3X knockout (DDX3X-KO) MEC-1 and JVM-3 cells. DDX3X-KO cells showed significantly increased proliferation and reduced spontaneous apoptosis compared to vector control. Additionally, DDX3X-KO cells showed higher rates of cell viability and reduced apoptosis after treatment of ibrutinib or venetoclax. We further found that DDX3X-KO cells had higher expression level of phosphorylated BTK and MCL-1, which may explain the resistance to ibrutinib and venetoclax, respectively. Moreover, DDX3X-KO cells showed increased expression level of CXCR4 and enhanced chemotaxis in response to CXCL-12. In summary, loss of DDX3X function promotes progression of CLL by mediating more invasive biological behaviors of CLL cells. To evaluate the mechanisms by which DDX3X deletion affects CLL, we detected key signaling pathways in the tumorigenesis and development of CLL in DDX3X-KO and control cells. We found increased expression of NOTCH1 and its downstream target proteins in DDX3X-KO cells (Figure 1), indicating that loss of DDX3X activated the NOTCH1 pathway. Considering that DDX3X is an RNA-binding protein and mainly regulates translation, we speculated that DDX3X may regulate the translation of NOTCH1. We performed RNA binding protein immunoprecipitation assay and found that DDX3X directly interacted with NOTCH1 mRNA (Figure 2A, B). Dual-luciferase reporter assay showed enhanced activity of NOTCH1 5‘ UTR in DDX3X-KO cells (Figure 2C), further suggesting that DDX3X may regulate translation of NOTCH1 mRNA through binding to the 5‘ UTR. Finally, polysome profiling demonstrated that DDX3X knockout resulted in increased NOTCH1 mRNAs in polysome fractions (Figure 2D), confirming that loss of DDX3X function facilitates NOTCH1 mRNA translation. In conclusion, DDX3X abnormalities are common in CLL and associated with poor prognosis. DDX3X dysregulation is involved in the progression of CLL by facilitating NOTCH1 mRNA translation through binding to the 5' UTR.