Endogenous Protein Research Articles

Structures, mechanisms and applications of RNA-centric CRISPR-Cas13.

Prokaryotes are equipped with a variety of resistance strategies to survive frequent viral attacks or invading mobile genetic elements. Among these, CRISPR-Cas surveillance systems are abundant and have been studied extensively. This Review focuses on CRISPR-Cas type VI Cas13 systems that use single-subunit RNA-guided Cas endonucleases for targeting and subsequent degradation of foreign RNA, thereby providing adaptive immunity. Notably, distinct from single-subunit DNA-cleaving Cas9 and Cas12 systems, Cas13 exhibits target RNA-activated substrate RNase activity. This Review outlines structural, biochemical and cell biological studies toward elucidation of the unique structural and mechanistic principles underlying surveillance effector complex formation, precursor CRISPR RNA (pre-crRNA) processing, self-discrimination and RNA degradation in Cas13 systems as well as insights into suppression by bacteriophage-encoded anti-CRISPR proteins and regulation by endogenous accessory proteins. Owing to its programmable ability for RNA recognition and cleavage, Cas13 provides powerful RNA targeting, editing, detection and imaging platforms with emerging biotechnological and therapeutic applications.

Abstract PO4-24-06: The function of GATA3-PARP1 complex in breast cancer during GATA3-mediated cellular reprogramming

Abstract In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins. Chromatin acts as a physical barrier for many transcription factors, preventing them from efficiently binding to their recognition sequences within the nucleosome. Pioneer factors are a class of transcription factors that bind to nucleosomes and activate silent chromatin. Pioneer factors are frequently involved in various immune cell pathways, tissue development, and homeostasis, thereby, underscoring their important roles in development and disease. Previous research found that pioneer factors have at least three fundamental properties: However, the molecular mechanisms of pioneer factor-mediated cellular reprogramming (mesenchymal-to-epithelial transition (MET)) are largely unknown. For this study, I will use GATA3-mediated MET to study the pioneer factor-mediated cellular reprogramming. GATA3 is a transcription factor that plays a pivotal role in mammary gland development, luminal epithelial differentiation, and cellular reprogramming. Additionally, it has been implicated in various cellular processes such as proliferation, migration, and invasion and is a critical regulator in breast cancer, including triple-negative breast cancer (TNBC). In TNBC, GATA3 expression is often downregulated, correlating with poor prognosis. Our lab and others have shown that GATA3 functions as a pioneer factor that actively changes the chromatin state from closed to open. In luminal breast cancer cells, ER-alpha and FOXA1 are well-known GATA3 co-factors. In mesenchymal breast cancer cells, GATA3 can suppress tumor metastasis by inducing MET in the absence of well-known GATA3 co-factors FOXA1 and ER-alpha1. These findings suggest that GATA3 works with additional, unknown co-factors during MET. We have been studying how GATA3 activates silent chromatin. More than 7 million GATA3 motifs exist in the human genome, yet the experimental data from GATA3 ChIP-seq analysis indicates less than 1% of the motifs are occupied by the pioneer factor, GATA3. In addition to binding selectivity, we have shown that the pioneer factor action is site-specific (context-dependent) and only induces chromatin opening and enhancer formation at a subset of binding sites. To identify a novel co-factor that is involved in GATA3-mediated MET, we performed a rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) assay, which identified the chromatin-modification enzyme, Poly ADP-ribose polymerase 1 (PARP1), as a potential GATA3 co-factor. PARP1 is essential for gene expression regulation and initiating DNA repair. Previous studies have shown PARP1 as a co-factor to the pioneer factor, SOX2. Our genomics data strongly suggest that PARP1 is involved in such context-dependent action of GATA3. Our overall goal is to investigate GATA3-PARP1 interaction during MET to understand the role of pioneer factors in cellular reprogramming. To do so, we are dissecting the GATA2-PARP1 function in two distinct steps: Citation Format: Mikhala Cooper, Motoki Takaku. The function of GATA3-PARP1 complex in breast cancer during GATA3-mediated cellular reprogramming [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-06.

Generating CRISPR Alleles in Drosophila.

CRISPR gene editing is a versatile and efficient approach for generating a wide variety of genetic reagents in flies. This unparalleled ability to manipulate the genome has revolutionized neuroscience, allowing Drosophila neurobiologists to readily generate new alleles to probe gene function, investigate the functional consequences of disease-associated variants, tag endogenous proteins to follow their dynamic localization in neurons and glia, and much more. Here, we provide a comprehensive protocol for generating heritable mutations in Drosophila We particularly focus on design considerations and tips for avoiding common errors to maximize the likelihood of successful gene editing.

Translational two-pore PBPK model to characterize whole-body disposition of different-size endogenous and exogenous proteins.

Two-pore physiologically based pharmacokinetic (PBPK) modeling has demonstrated its potential in describing the pharmacokinetics (PK) of different-size proteins. However, all existing two-pore models lack either diverse proteins for validation or interspecies extrapolation. To fill the gap, here we have developed and optimized a translational two-pore PBPK model that can characterize plasma and tissue disposition of different-size proteins in mice, rats, monkeys, and humans. Datasets used for model development include more than 15 types of proteins: IgG (150kDa), F(ab)2 (100kDa), minibody (80kDa), Fc-containing proteins (205, 200, 110, 105, 92, 84, 81, 65, or 60kDa), albumin conjugate (85.7kDa), albumin (67kDa), Fab (50kDa), diabody (50kDa), scFv (27kDa), dAb2 (23.5kDa), proteins with an albumin-binding domain (26, 23.5, 22, 16, 14, or 13kDa), nanobody (13kDa), and other proteins (110, 65, or 60kDa). The PBPK model incorporates: (i) molecular weight (MW)-dependent extravasation through large and small pores via diffusion and filtration, (ii) MW-dependent renal filtration, (iii) endosomal FcRn-mediated protection from catabolism for IgG and albumin-related modalities, and (iv) competition for FcRn binding from endogenous IgG and albumin. The finalized model can well characterize PK of most of these proteins, with area under the curve predicted within two-fold error. The model also provides insights into contribution of renal filtration and lysosomal degradation towards total elimination of proteins, and contribution of paracellular convection/diffusion and transcytosis towards extravasation. The PBPK model presented here represents a cross-modality, cross-species platform that can be used for development of novel biologics.

The Role of Renal Medullary Bilirubin and Biliverdin Reductase in Ang II Induced Hypertension

Previous studies have demonstrated the importance of renal medullary heme oxygenase-1 (HO-1) in blood pressure response to angiotensin II. However, the role of the HO metabolite, bilirubin, in this response remains unknown. The goal of the present study was to test the hypothesis that generation of bilirubin in the renal medulla plays an important role in the regulation of blood pressure in response to angiotensin II. Twenty-week-old male C57Bl/6J mice were implanted with intrarenal medullary interstitial (IRMI) catheters following unilateral nephrectomy. After this time, biliverdin IXα was specifically infused into the kidney (3.6 mg/day) for 3 days prior to implantation with an osmotic minipump delivering angiotensin II (1000 ng/kg/min). Blood pressure was recorded for 3 days, 1 week after minipump infusion, in conscious mice. IRMI infusion of biliverdin significantly decreased blood pressure as compared to mice infused with vehicle (118 ± 4 vs. 158 ± 2 mmHg, p<0.05). To further explore the antihypertensive role of renal medullary bilirubin generation, mice with specific deletion of biliverdin reductase-A (BVRA) in the thick ascending loop of Henle (TALH) were generated by crossing BVRAflox/flox mice with Tamm-Horsfall Cre (THP-Cre) mice. THP-Cre/BVRAflox/flox mice exhibited specific deletion of BVRA in the TALH by co-immunostaining with endogenous THP protein. THP-Cre/BVRAflox/flox mice did not exhibit any differences in baseline blood pressure as compared to BVRAflox/flox mice (108 ± 2 vs. 108 ± 2 mmHg). However, angiotensin-II infusion resulted in significantly higher blood pressure measured in conscious mice 7 days after implantation in THP-Cre/BVRAflox/flox as compared to BVRAflox/flox mice (152 ± 2 vs. 140 ± 3 mmHg, p<0.05). These results demonstrate an important role for renal medullary bilirubin and biliverdin reductase in the blood pressure response to angiotensin II. Strategies which increase renal medullary bilirubin/biliverdin reductase levels could be novel antihypertensive therapies. Future studies will examine the mechanistic link between bilirubin and electrolyte transporters in the kidney, specifically, the Na+-K+-2Cl2 cotransporter (NKCC2), which is a major transporter of electrolyte in the thick ascending limb and is known to play a role in Ang II-induced hypertension. This work was supported by National Institutes of Health grants (RO1-HL088421 to DS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Smooth Muscle Cofilin Confers Elasticity to Coronary Resistance Microvessels in Diabetes

Coronary resistance microvessels (CRMs) from type 2 diabetic (T2D) mice and pigs are less stiff compared to normal, a finding that is dictated by less stiff coronary vascular smooth muscle cells (VSMCs) Cofilin is an endogenous actin regulatory protein that depolymerizes filamentous F-actin, and recent studies have shown that portions of F-actin bound to cofilin are less stiff compared to their unbound F-actin counterparts. In this study, we tested the hypothesis that augmented cofilin confers elasticity to diabetic CRMs. CRMs were isolated and pooled (n=6 pooled CRMs per biological replicate) for proteomic data collection from both normal (n=3) and diabetic (n=5) mice. In separate experiments, we utilized deidentified normal and T2D human primary coronary VSMCs obtained from commercial vendors (Lonza and ATCC; n=4 per group) and experiments were performed on passage 5-8 VSMCs. Using these cells, we determined cofilin expression by Western blot, and cofilin siRNA (Cell Signaling Technology) was used 48 hours prior to experimentation to knock down cofilin expression. Elastic modulus, a measure of cell stiffness, was assessed by atomic force microscopy (AFM). Globular G-actin was labeled with fluorescein-deoxyribonuclease I (DNAse I) and F-actin was labeled with phalloidin. Images were taken in blinded manner and Image J was used to assess expression. In the proteomics screen, we observed zero cofilin spectral hits in the normal CRMs and 3-4 spectral hits in db/db CRMs (0.00 ± 0.0 vs. 3.40 ± 0.25, p<0.0001). Cofilin protein was increased in the coronary VSMCs of diabetic patients compared to normal coronary VSMCs (1.00 ± 0.16 vs. 1.837± 0.35, p=0.052). In human coronary VSMCs, silencing cofilin caused a significant increase in the F/G actin ratio when normal and diabetic data were pooled (scramble 3.029 ± 0.49 vs. siRNA 4.029 ± 0.68, p= 0.018, n=8). This increased F/G ratio was significant in the diabetic coronary VSMCs (scramble 2.636 ± 0.46 vs. siRNA 3.685 ± 0.41, p=0.049, n=4), and trended in the normal cells but did not reach statistical significance (scramble 3.423 ± 0.89 vs. cofilin siRNA 4.373 ± 1.37, p=0.224, n=4). Cofilin knockdown also caused a significant increase in elastic modulus by AFM when normal and diabetic data were pooled (scramble siRNA 1.814 ± 0.25 vs. cofilin siRNA 3.074 ± 0.35, p=0.0057, n=8). This trend persisted in the normal VMSCs (scramble siRNA 2.038 ± 0.31 vs. cofilin siRNA 3.445± 0.30, p=0.075, n=4) and in the diabetic VSMCs (scramble siRNA 1.589 ± 0.40 vs. cofilin siRNA 2.702 ± 0.62, p=0.085, n=4) but did not reach significance due to low available sample number. Overall, we demonstrate increased cofilin in human diabetic coronary VSMCs and in CRMs from diabetic mice, and silencing cofilin increased F/G actin ratio and increased cellular stiffness. These data support the notion that increased cofilin in T2D VSMCs may be the cause of decreased diabetic CRM stiffness. Support: NIH R00 HL116769, R01 HL165124, and S10 OD023438 and Nationwide Children’s Hospital to AJT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BCL6 is a context-dependent mediator of the glioblastoma response to irradiation therapy

Glioblastoma is a rapidly fatal brain cancer that does not respond to therapy. Previous research showed that the transcriptional repressor protein BCL6 is upregulated by chemo and radiotherapy in glioblastoma, and inhibition of BCL6 enhances the effectiveness of these therapies. Therefore, BCL6 is a promising target to improve the efficacy of current glioblastoma treatment. BCL6 acts as a transcriptional repressor in germinal centre B cells and as an oncogene in lymphoma and other cancers. However, in glioblastoma, BCL6 induced by therapy may not be able to repress transcription. Using a BCL6 inhibitor, the whole proteome response to irradiation was compared with and without BCL6 activity. Acute high dose irradiation caused BCL6 to switch from repressing the DNA damage response to promoting stress response signalling. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) enabled comparison of BCL6 partner proteins between untreated and irradiated glioblastoma cells. BCL6 was associated with transcriptional coregulators in untreated glioblastoma including the known partner NCOR2. However, this association was lost in response to acute irradiation, where BCL6 unexpectedly associated with synaptic and plasma membrane proteins. These results reveal the activity of BCL6 under therapy-induced stress is context-dependent, and potentially altered by the intensity of that stress.

Multiple strategies to improve the secretory expression of prolyl endopeptidase in Aspergillus niger

Prolyl Endopeptidase (PEP) has the unique ability to hydrolyze the carboxyl terminal peptide bonds of Proline. This enzyme holds great value in various fields including medical research, disease treatment, human diet, and food production. Aspergillus niger is a significant food-grade protein expression system renowned for its high safety. It is considered an ideal system for producing Aspergillus-derived PEP due to its ability to secrete a large amount of endogenous proteins. Our research group successfully constructed the Aspergillus niger PEP engineering strain in the early stages by eliminating the background protein AsaA, thereby increasing secretion protein production. In this article, we explore new solutions to further enhance production. Experimental investigations were conducted to regulate secretory protein production at different levels by modifying the 5′UTR and reconstructing the secretion pathways. The results demonstrate that the strain modified by the 5′UTR and Kozak sequence (glaA5'UTR-GCCACC, glaA::protA) exhibited a 1.88-fold increase in gene transcription and a 105% increase in extracellular PEP activity compared to the non-modified strain. Co-expression of the ERdjs gene AnScj1 further increased extracellular PEP activity by 168%. However, deletion of derA or vps10, key genes in the Aspergillus niger secretory protein degradation pathway, resulted in reduced levels of gene transcription and secretory expression. This article holds practical significance in enhancing the secretion and expression of PEP, obtaining higher-yield strains, and exploring optimization strategies for highly expressed proteins.

PKIB, a Novel Target for Cancer Therapy.

The serine-threonine kinase protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent intracellular protein with multiple roles in cellular biology including metabolic and transcription regulation functions. The cAMP-dependent protein kinase inhibitor β (PKIB) is one of three known endogenous protein kinase inhibitors of PKA. The role of PKIB is not yet fully understood. Hormonal signaling is correlated with increased PKIB expression through genetic regulation, and increasing PKIB expression is associated with decreased cancer patient prognosis. Additionally, PKIB impacts cancer cell behavior through two mechanisms; the first is the nuclear modulation of transcriptional activation and the second is the regulation of oncogenic AKT signaling. The limited research into PKIB indicates the oncogenic potential of PKIB in various cancers. However, some studies suggest a role of PKIB in non-cancerous disease states. This review aims to summarize the current literature and background of PKIB regarding cancer and related issues. In particular, we will focus on cancer development and therapeutic possibilities, which are of paramount interest in PKIB oncology research.

Extensive protein pyrophosphorylation revealed in human cell lines

Reversible protein phosphorylation is a central signaling mechanism in eukaryotes. Although mass-spectrometry-based phosphoproteomics has become routine, identification of non-canonical phosphorylation has remained a challenge. Here we report a tailored workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing, to our knowledge, the first direct evidence of endogenous protein pyrophosphorylation. We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. Detection was consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). When the biosynthesis of PP-InsPs was perturbed, proteins expressed in this background exhibited no signs of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1 and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation and should be considered in future phosphoproteomic analyses.

Force-Based Neuromodulation.

Technologies for neuromodulation have rapidly developed in the past decade with a particular emphasis on creating noninvasive tools with high spatial and temporal precision. The existence of such tools is critical in the advancement of our understanding of neural circuitry and its influence on behavior and neurological disease. Existing technologies have employed various modalities, such as light, electrical, and magnetic fields, to interface with neural activity. While each method offers unique advantages, many struggle with modulating activity with high spatiotemporal precision without the need for invasive tools. One modality of interest for neuromodulation has been the use of mechanical force. Mechanical force encapsulates a broad range of techniques, ranging from mechanical waves delivered via focused ultrasound (FUS) to torque applied to the cell membrane.Mechanical force can be delivered to the tissue in two forms. The first form is the delivery of a mechanical force through focused ultrasound. Energy delivery facilitated by FUS has been the foundation for many neuromodulation techniques, owing to its precision and penetration depth. FUS possesses the potential to penetrate deeply (∼centimeters) into tissue while maintaining relatively precise spatial resolution, although there exists a trade-off between the penetration depth and spatial resolution. FUS may work synergistically with ultrasound-responsive nanotransducers or devices to produce a secondary energy, such as light, heat, or an electric field, in the target region. This layered technology, first enabled by noninvasive FUS, overcomes the need for bulky invasive implants and also often improves the spatiotemporal precision of light, heat, electrical fields, or other techniques alone. Conversely, the second form of mechanical force modulation is the generation of mechanical force from other modalities, such as light or magnetic fields, for neuromodulation via mechanosensitive proteins. This approach localizes the mechanical force at the cellular level, enhancing the precision of the original energy delivery. Direct interaction of mechanical force with tissue presents translational potential in its ability to interface with endogenous mechanosensitive proteins without the need for transgenes.In this Account, we categorize force-mediated neuromodulation into two categories: 1) methods where mechanical force is the primary stimulus and 2) methods where mechanical force is generated as a secondary stimulus in response to other modalities. We summarize the general design principles and current progress of each respective approach. We identify the key advantages of the limitations of each technology, particularly noting features in spatiotemporal precision, the need for transgene delivery, and the potential outlook. Finally, we highlight recent technologies that leverage mechanical force for enhanced spatiotemporal precision and advanced applications.

Bovine placental extracellular vesicles carry the fusogenic syncytin BERV-K1

Syncytins are endogenous retroviral envelope proteins which induce the fusion of membranes. A human representative of this group, endogenous retrovirus group W member 1 envelope (ERVW-1) or syncytin-1 is present in trophoblast-derived extracellular vesicles and supports the incorporation of these extracellular vesicles into recipient cells. During pregnancy, placenta-derived extracellular vesicles participate in feto-maternal communication. Bovine fetal binucleate trophoblast cells express the syncytin, bovine endogenous retroviral envelope protein K1 (BERV-K1). These cells release extracellular vesicles into the maternal stroma, but it is unclear whether BERV-K1 is included in these extracellular vesicles. Here, extracellular vesicles were isolated from bovine placental tissue using collagenase digestion, ultracentrifugation, and size exclusion chromatography. They were characterized with transmission electron microscopy, nanoparticle tracking analysis, immunoblotting and mass spectrometry. Immunohistochemistry and immunoelectron microscopy were used to localize BERV-K1 within the bovine placental tissue. The isolated extracellular vesicles range between 50 and 300 nm, carrying multiple extracellular vesicle biomarkers. Proteomic analysis and immunoelectron microscopy confirmed BERV-K1 presence on the isolated extracellular vesicles. Further, BERV-K1 was localized on intraluminal vesicles in secretory granules of binucleate trophoblast cells. The presence of BERV-K1 on bovine placental extracellular vesicles suggests their role in feto-maternal communication and potential involvement of BERV-K1 in uptake of extracellular vesicles by target cells.

Cross-Reactivity of N6AMT1 Antibodies with Aurora Kinase A: An Example of Antibody-Specific Non-Specificity.

Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with endogenous and recombinant mitosis-associated protein Aurora kinase A (AURKA). The cross-reactivity was verified through immunofluorescence, immunoblot, and immunoprecipitation assays combined with mass spectrometry. N6AMT1 and AURKA are evolutionarily conserved proteins that are vital for cellular processes. Both proteins share the motif ENNPEE, which is unique to only these two proteins. We suggest that N6AMT1 antibodies recognise this motif in N6AMT1 and AURKA proteins and exhibit an example of "specific" non-specificity. This serves as an example of the importance of controls and critical data interpretation in molecular biology research.

The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

Porcine epidemic diarrhea virus E protein induces formation of stress granules and attenuates protein translation through activation of the PERK/eIF2α signaling pathway

Porcine epidemic diarrhea virus (PEDV) envelope protein (E) has been characterized as an important structural protein that plays critical roles in the interplay with its host to affect the virus life cycle. Stress granules (SGs) are host translationally silent ribonucleoproteins, which are mainly induced by the phosphorylation of eIF2α in the PERK/eIF2α signaling pathway. Our previous study found that PEDV E protein caused endoplasmic reticulum stress response (ERS)-mediated suppression of antiviral proteins’ translation. However, the link and the underlying mechanism by which PEDV induces SGs formation and suppresses host translation remain elusive. In this study, our results showed that PEDV E protein significantly elevated the expression of GRP78, CANX, and phosphorylation of PERK and eIF2α, indicating that the PERK/eIF2α branch of ERS was activated. PEDV E protein localized to the ER and aggregated into puncta to reconstruct ER structure, and further induced SGs formation, which has been caused through upregulating the G3BP1 expression level. In addition, a significant global translational stall and endogenous protein translation attenuation were detected in the presence of E protein overexpression, but the global mRNA transcriptional level remained unchanged, suggesting that the shutoff of protein translation was associated with the translation, not with the transcription process. Collectively, this study demonstrates that PERK/eIF2α activation is required for SGs formation and protein translation stall. This study is beneficial for us to better understand the mechanism by which PEDV E suppresses host protein synthesis, and provides us a new insight into the host translation regulation during virus infection.

Antioxidant and anti‑inflammatory effects of esculin and esculetin (Review).

Fraxinus chinensis Roxb is a deciduous tree, which is distributed worldwide and has important medicinal value. In Asia, the bark of Fraxinus chinensis Roxb is a commonly used traditional Chinese medicine called Qinpi. Esculetin is a coumarin compound derived from the bark of Fraxinus chinensis Roxb and its glycoside form is called esculin. The aim of the present study was to systematically review relevant literature on the antioxidant and anti-inflammatory effects of esculetin and esculin. Esculetin and esculin can promote the expression of various endogenous antioxidant proteins, such as superoxide dismutase, glutathione peroxidase and glutathione reductase. This is associated with the activation of the nuclear factor erythroid-derived factor 2-related factor 2 signaling pathway. The anti-inflammatory effects of esculetin and esculin are associated with the inhibition of the nuclear factor κ-B and mitogen-activated protein kinase inflammatory signaling pathways. In various inflammatory models, esculetin and esculin can reduce the expression levels of various proinflammatory factors such as tumor necrosis factor-α, interleukin (IL)-1β and IL-6, thereby inhibiting the development of inflammation. In summary, esculetin and esculin may be promising candidates for the treatment of numerous diseases associated with inflammation and oxidative stress, such as ulcerative colitis, acute lung and kidney injury, lung cancer, acute kidney injury.

Characterization of the endogenous retrovirus-derived placenta-specific soluble protein EnvV-Fca from domestic cats.

Endogenous retroviruses (ERVs) are remnants of ancestral viruses in the host genome. The present study identified the expression of a defective retroviral env gene belonging to the ERV group V member Env (EnvV) in Felis catus (EnvV-Fca). EnV-Fca was specifically detected in the placental trophoblast syncytiotrophobic layer and expressed as a secreted protein in cultured cells. Genetic analyses indicated that EnvV2 genes are widely present in vertebrates and are under purifying selection among carnivores, suggesting a potential benefit for the host. This study suggests that birds, bats, and rodents carrying EnvV2 may play significant roles as intermediate vectors in spreading or cross-transmitting viruses among species. Our findings provide valuable insights into the evolution of ERV in vertebrate hosts.

Protective Effects of Coptis chinensis Rhizome Extract and Its Constituents (Berberine, Coptisine, and Palmatine) against α-Synuclein Neurotoxicity in Dopaminergic SH-SY5Y Cells.

Parkinson's disease (PD) is a common neurodegenerative disease with progressive loss of dopaminergic neurons in substantia nigra and the presence of α-synuclein-immunoreactive inclusions. Gaucher's disease is caused by homozygous mutations in β-glucocerebrosidase gene (GBA). GBA mutation carriers have an increased risk of PD. Coptis chinensis (C. chinensis) rhizome extract is a major herb widely used to treat human diseases. This study examined the association of GBA L444P mutation with Taiwanese PD in 1016 cases and 539 controls. In addition, the protective effects of C. chinensis rhizome extract and its active constituents (berberine, coptisine, and palmatine) against PD were assayed using GBA reporter cells, LC3 reporter cells, and cells expressing mutated (A53T) α-synuclein. Case-control study revealed that GBA L444P carriers had a 3.93-fold increased risk of PD (95% confidence interval (CI): 1.37-11.24, p = 0.006) compared to normal controls. Both C. chinensis rhizome extract and its constituents exhibited chemical chaperone activity to reduce α-synuclein aggregation. Promoter reporter and endogenous GBA protein analyses revealed that C. chinensis rhizome extract and its constituents upregulated GBA expression in 293 cells. In addition, C. chinensis rhizome extract and its constituents induced autophagy in DsRed-LC3-expressing 293 cells. In SH-SY5Y cells expressing A53T α-synuclein, C. chinensis rhizome extract and its constituents reduced α-synuclein aggregation and associated neurotoxicity by upregulating GBA expression and activating autophagy. The results of reducing α-synuclein aggregation, enhancing GBA expression and autophagy, and protecting against α-synuclein neurotoxicity open up the therapeutic potentials of C. chinensis rhizome extract and constituents for PD.

TANK Binding Kinase 1 Promotes BACH1 Degradation through Both Phosphorylation-Dependent and -Independent Mechanisms without Relying on Heme and FBXO22.

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.

Unleashing Natural IL18 Activity Using an Anti-IL18BP Blocker Induces Potent Immune Stimulation and Antitumor Effects.

Recombinant cytokines have limited anticancer efficacy mostly due to a narrow therapeutic window and systemic adverse effects. IL18 is an inflammasome-induced proinflammatory cytokine, which enhances T- and NK-cell activity and stimulates IFNγ production. The activity of IL18 is naturally blocked by a high-affinity endogenous binding protein (IL18BP). IL18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. In this study, we found that IL18 is upregulated in the TME compared with the periphery across multiple human tumors and most of it is bound to IL18BP. Bound IL18 levels were largely above the amount required for T-cell activation in vitro, implying that releasing IL18 in the TME could lead to potent T-cell activation. To restore the activity of endogenous IL18, we generated COM503, a high-affinity anti-IL18BP that blocks the IL18BP:IL18 interaction and displaces precomplexed IL18, thereby enhancing T- and NK-cell activation. In vivo, administration of a surrogate anti-IL18BP, either alone or in combination with anti-PD-L1, resulted in significant tumor growth inhibition and increased survival across multiple mouse tumor models. Moreover, the anti-IL18BP induced pronounced TME-localized immune modulation including an increase in polyfunctional nonexhausted T- and NK-cell numbers and activation. In contrast, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, blocking IL18BP using an Ab is a promising approach to harness cytokine biology for the treatment of cancer.