Mini PROTACs: N-end rule-mediated degradation on the horizon.

Heterobifunctional proteolysis-targeting chimeras (PROTACs) offer a promising cancer treatment avenue by efficiently degrading unwanted cellular proteins. A recent study from Zhang et al. demonstrated the successful utilization of the N-end rule in PROTAC design, allowing for a modular degradation rate tailored to the oncogenic driver BCR-ABL.

Metagenomic next-generation sequencing for rapid detection of pulmonary infection in patients with acquired immunodeficiency syndrome.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) is associated with a high rate of pulmonary infections (bacteria, fungi, and viruses). To overcome the low sensitivity and long turnaround time of traditional laboratory-based diagnostic strategies, we adopted metagenomic next-generation sequencing (mNGS) technology to identify and classify pathogens. RESULTS: This study enrolled 75 patients with AIDS and suspected pulmonary infections who were admitted to Nanning Fourth People's Hospital. Specimens were collected for traditional microbiological testing and mNGS-based diagnosis. The diagnostic yields of the two methods were compared to evaluate the diagnostic value (detection rate and turn around time) of mNGS for infections with unknown causative agent. Accordingly, 22 cases (29.3%) had a positive culture and 70 (93.3%) had positive valve mNGS results (P value < 0.0001, Chi-square test). Meanwhile, 15 patients with AIDS showed concordant results between the culture and mNGS, whereas only one 1 patient showed concordant results between Giemsa-stained smear screening and mNGS. In addition, mNGS identified multiple microbial infections (at least three pathogens) in almost 60.0% of patients with AIDS. More importantly, mNGS was able to detect a large variety of pathogens from patient tissue displaying potential infection and colonization, while culture results remained negative. There were 18 members of pathogens which were consistently detected in patients with and without AIDS. CONCLUSIONS: In conclusion, mNGS analysis provides fast and precise pathogen detection and identification, contributing substantially to the accurate diagnosis, real-time monitoring, and treatment appropriateness of pulmonary infection in patients with AIDS.

Ribavirin inhibits peste des petits ruminants virus proliferation in vitro.

Peste des petits ruminants virus (PPRV), a member of the family Paramyxoviridae, belongs to the genus Morbillivirus. It causes devastating viral diseases in small ruminants and has been rapidly spreading over various regions in Africa, the Middle East, and Asia. Although vaccination is thought to be an effective management strategy against PPR infections, the heat sensitivity of PPRV vaccines severely restricts their use in regions with hot climates. In this research, we studied the antiviral activities of ribavirin and aimed to understand the potential mechanisms of action of ribavirin in the African green monkey kidney cells (Vero cells). In brief, the adsorption, intrusion, replication, and release of PPRV, as well as the mRNA expression level of RNA-dependent RNA polymerase (RdRp), were significantly inhibited in the ribavirin-treated Vero cells compared to those in the PPRV-infected cells that were not treated with ribavirin. Additionally, ribavirin has potential as an antiviral drug against PPRV, and its antiviral activity is mediated by the Janus kinase signal transducer and activator of transcription (JAK/STAT) and PI3K/AKT pathways.

CXCL9-modified CAR T cells improve immune cell infiltration and antitumor efficacy.

Chimeric antigen receptor (CAR) T cells remain unsatisfactory in treating solid tumors. The frequency of tumor-infiltrating T cells is closely related to the good prognosis of patients. Augmenting T cell accumulation in the tumor microenvironment is essential for tumor clearance. To overcome insufficient immune cell infiltration, innovative CAR designs need to be developed immediately. CXCL9 plays a pivotal role in regulating T cell migration and inhibiting tumor angiogenesis. Therefore, we engineered CAR T cells expressing CXCL9 (CART-CXCL9). The addition of CXCL9 enhanced cytokine secretion and cytotoxicity of CAR T cells and endowed CAR T cells with the ability to recruit activated T cells and antiangiogenic effect. In tumor-bearing mice, CART-CXCL9 cells attracted more T cell trafficking to the tumor site and inhibited angiogenesis than conventional CAR T cells. Additionally, CART-CXCL9 cell therapy slowed tumor growth and prolonged mouse survival, displaying superior antitumor activity. Briefly, modifying CAR T cells to express CXCL9 could effectively improve CAR T cell efficacy against solid tumors.

Long-term clinical efficacy of cytokine-induced killer cell-based immunotherapy in early-stage esophageal squamous cell carcinoma.

BACKGROUND AIMS: In this retrospective clinical study, the authors investigated the impact of cytokine-induced killer (CIK) cell-based immunotherapies on the long-term survival of patients with esophageal squamous cell carcinoma (ESCC). METHODS: A total of 87 patients with ESCC who received comprehensive treatment were enrolled in the study. Of these patients, 43 were in the control group and 44 were in the CIK treatment group. Flow cytometry analysis was performed to detect the phenotype and anti-tumor function of CIK cells. Clinical characteristics were compared between these two groups, and the survival estimates of ESCC patients were determined using Kaplan-Meier analysis. RESULTS: CIK cells contained a high proportion of the main functional fraction (CD3+CD56+ group) and exhibited a strong killing ability for esophageal cancer cells in vitro. Importantly, overall survival (OS) and progression-free survival (PFS) were significantly higher in the CIK group than in the control group in early-stage ESCC. However, patients with advanced-stage ESCC did not benefit from CIK cell-based therapy in terms of OS and PFS compared with the control group. CONCLUSIONS: These results demonstrate that CIK cells combined with conventional treatments potentially prolong long-term survival of patients and may serve as a combined therapeutic approach for the treatment of early-stage ESCC.

In vivo biological safety evaluation of an iron-based bioresorbable drug-eluting stent.

Bioresorbable stents (BRS) are investigated and designed to revascularize occluded arteries. The iron-based bioresorbable stent is a promising device in interventional therapies, although it's corrosion and bioresorption rate remain challenging. In this work, we introduced a novel nitrided iron coronary stent (IBS) with enhanced degradation rate compared with pure iron stent. To evaluate the biosafety of this device, a sub-chronic systemic toxicity study was conducted and a stainless steel stent (Supporter™) served as a control. Here, the bioresorbable stent was first evaluated in rat abdominal aortic implantation model. When subjected to exaggerated exposure dose, no clinical signs of toxicity or mortality were observed in either, the IBS group or the control group. Histopathological examinations showed the corrosion particles of iron were encapsulated by fibrocytes and engulfed by macrophages, indicating that the degradation of iron was in the early stage. Our results demonstrated that the nitrided iron stent did not induce systemic toxicity under the experimental conditions.

A ubiquitin ligase-associated chaperone holdase maintains polypeptides in soluble states for proteasome degradation.

Endoplasmic reticulum-associated degradation (ERAD) employs membrane-bound ubiquitin ligases and the translocation-driving ATPase p97 to retrotranslocate misfolded proteins for proteasomal degradation. How retrotranslocated polypeptides bearing exposed hydrophobic motifs or transmembrane domains (TMDs) avoid aggregation before reaching the proteasome is unclear. Here we identify a ubiquitin ligase-associated multiprotein complex comprising Bag6, Ubl4A, and Trc35, which chaperones retrotranslocated polypeptides en route to the proteasome to improve ERAD efficiency. In vitro, Bag6, the central component of the complex, contains a chaperone-like activity capable of maintaining an aggregation-prone substrate in an unfolded yet soluble state. The physiological importance of this holdase activity is underscored by observations that ERAD substrates accumulate in detergent-insoluble aggregates in cells depleted of Bag6, or of Trc35, a cofactor that keeps Bag6 outside the nucleus for engagement in ERAD. Our results reveal a ubiquitin ligase-associated holdase that maintains polypeptide solubility to enhance protein quality control in mammalian cells.

Ubiquitin C-Terminal Hydrolase L1 regulates autophagy by inhibiting autophagosome formation through its deubiquitinating enzyme activity.

Ubiquitination modification has been shown to play a key role in autophagy. Increasing studies reported the involvement of de-ubiquitinating enzymes (DUBs) in autophagy pathway. To systematically search how DUBs manipulate autophagy, we utilized a double fluorescence tagged LC3 stable HeLa cell line, and did a genome wide screen of 55 human DUBs which is about 60% coverage of the DUB family. We found a bunch of DUBs have impact on autophagy by either changing the LC3 puncta formation or the autophagy flux. One of them, Ubiquitin C-Terminal Hydrolase L1 (UCHL1) correlated to Parkinson's disease, strongly affects autophagy by inhibiting autophagosome formation. We found UCHL1 overexpression inhibits LC3 puncta formation and is dependent on its DUB activity. Knockdown of UCHL1 significantly promotes LC3 puncta formation. Further study revealed that UCHL1 may affect autophagy by interacting with LC3 but not other autophagy related proteins. Interestingly, a Parkinson's disease related mutant UCHL1 I93 M defects its DUB activity and can no longer inhibit autophagosome formation. We further screened 22 commercially available DUB inhibitors and found two potent UCHL1 inhibitors LDN-57444 (LDN) and NSC632839 (NSC), when treating cells, both strongly induce LC3 puncta formation. Taken together, our results indicated a new insight into the manner in which DUB regulates autophagy and provided potential drugs for the Parkinson's disease.

A genetics laboratory class to analyze early and late feather traits of chicken.

With the implementation of the "Education and Training Program for Outstanding Agricultural and Forestry Talents" in our country, our university established the "Outstanding Class" for students majoring in the animal science. We also carried out a series of educational management and curriculum reforms to cultivate students' systematic model of thinking and the ability of technology innovation. In this paper, we designed a comprehensive experiment that focused on analyzing early and late feather genetic traits of chicken. The students initially observed the phenotype of chickens and gradually were led into genetics analysis. We introduced the breeding practice, and guided the students to use genetic theories to breed chick strains of early and late feather traits. The experiment is not only based on the sex-linkage theory and sex determination mechanism, but also molecular genetics technologies, such as genomic DNA extraction, amplification, enzyme digestion and electrophoresis. Conducting this experiment can enhance students' comprehensive analysis ability and professional skills, as well as be beneficial to cultivate their scientific research interests and curiosity on animal sciences. Thus, we integrated the genetics theories into animal breeding practice that meet the requirement of comprehensive applied talents of animal science specialty. The teaching ideas and methods described in this paper can be applied to other biological experiment teaching practice.

Lunapark Is a Component of a Ubiquitin Ligase Complex Localized to the Endoplasmic Reticulum Three-way Junctions.

The endoplasmic reticulum (ER) network comprises sheets and tubules that are connected by dynamic three-way junctions. Lunapark (Lnp) localizes to and stabilizes ER three-way junctions by antagonizing the small GTPase Atlastin, but how Lnp shapes the ER network is unclear. Here, we used an affinity purification approach and mass spectrometry to identify Lnp as an interacting partner of the ER protein quality control ubiquitin ligase gp78. Accordingly, Lnp purified from mammalian cells has a ubiquitin ligase activity in vitro Intriguingly, biochemical analyses show that this activity can be attributed not only to associated ubiquitin ligase, but also to an intrinsic ubiquitin ligase activity borne by Lnp itself. This activity is contained in the N-terminal 45 amino acids of Lnp although this segment does not share homology to any known ubiquitin ligase motifs. Despite its interaction with gp78, Lnp does not seem to have a broad function in degradation of misfolded ER proteins. On the other hand, the N-terminal ubiquitin ligase-bearing motif is required for the ER three-way junction localization of Lnp. Our study identifies a new type of ubiquitin ligase and reveals a potential link between ubiquitin and ER morphology regulation.

Homocysteine, Ischemic Stroke, and Coronary Heart Disease in Hypertensive Patients: A Population-Based, Prospective Cohort Study.

BACKGROUND AND PURPOSE: Total homocysteine level (tHcy) is a risk factor of ischemic stroke (IS) and coronary heart disease. However, the results are conflicting and mainly focused on healthy individuals in developed countries. METHODS: A prospective, population-based cohort study was conducted among 5935 participants from 60 communities in the city of Shenzhen, China. A Cox regression analysis was applied to evaluate the contribution of tHcy to the risk of IS and coronary heart disease. The effect of folic acid supplementation on tHcy levels was also evaluated among 501 patients with essential hypertension, who received an average of 2.5 years of folic acid supplementation. RESULTS: After adjustment for confounding factors, the hazard ratios (95% confidence intervals) of IS caused by hyperhomocysteinemia were 2.18 (1.65-2.89), 2.40 (1.56-3.67), and 2.73 (1.83-4.08) in the total, male, and female participants, respectively. Compared with normal levels of tHcy (<15 µmol/L), the hazard ratios (95% confidence intervals) for IS in the highest tHcy category (≥30 µmol/L) were 4.96 (3.03-8.12), 6.11 (3.44-10.85), and 1.84 (0.52-6.46) in the total, males, and females participants, respectively. However, we did not observe a significant relationship between tHcy and the risk of coronary heart disease. The 2.5 years of folic acid supplementation reduced tHcy levels by 6.7 µmol/L (27.92%) in patients with essential hypertension. CONCLUSIONS: Hyperhomocysteinemia in Chinese hypertensive patients is significantly associated with IS risk but not coronary heart disease susceptibility, and folic acid supplementation can efficiently reduce tHcy levels.

Ubiquitin-specific protease16 modulates salt tolerance in Arabidopsis by regulating Na(+)/H(+) antiport activity and serine hydroxymethyltransferase stability.

Protein ubiquitination is a reversible process catalyzed by ubiquitin ligases and ubiquitin-specific proteases (UBPs). We report the identification and characterization of UBP16 in Arabidopsis thaliana. UBP16 is a functional ubiquitin-specific protease and its enzyme activity is required for salt tolerance. Plants lacking UBP16 were hypersensitive to salt stress and accumulated more sodium and less potassium. UBP16 positively regulated plasma membrane Na(+)/H(+) antiport activity. Through yeast two-hybrid screening, we identified a putative target of UBP16, SERINE HYDROXYMETHYLTRANSFERASE1 (SHM1), which has previously been reported to be involved in photorespiration and salt tolerance in Arabidopsis. We found that SHM1 is degraded in a 26S proteasome-dependent process, and UBP16 stabilizes SHM1 by removing the conjugated ubiquitin. Ser hydroxymethyltransferase activity is lower in the ubp16 mutant than in the wild type but higher than in the shm1 mutant. During salt stress, UBP16 and SHM1 function in preventing cell death and reducing reactive oxygen species accumulation, activities that are correlated with increasing Na(+)/H(+) antiport activity. Overexpression of SHM1 in the ubp16 mutant partially rescues its salt-sensitive phenotype. Taken together, our results suggest that UBP16 is involved in salt tolerance in Arabidopsis by modulating sodium transport activity and repressing cell death at least partially through modulating SMH1stability and activity.

Association between homocysteine and incidence of ischemic stroke in subjects with essential hypertension: a matched case-control study.

BACKGROUND: To assess the association between total plasma homocysteine (tHcy) and ischemic stroke (IS) in hypertensive subjects in a matched case-control study. METHODS: This is a 1:2 matched and population-based case-control study, all of the participants were recruited from the 60 communities in Shenzhen, China. Demographic and socioeconomic characteristics, medical records, lifestyle risk factors and other clinical characteristics were obtained from all of the subjects. The association between tHcy and incidence of IS was analyzed by using conditional logistic regression models. RESULTS: The median values of plasma tHcy were significantly higher in IS subjects than in non-IS subjects, especially in women. After adjusted for the confounding factors in Model 2, compared with the lowest quartile of tHcy, the odds ratios (ORs) and 95% CIs of the highest quartile of tHcy for IS were 0.83 (0.36-1.90) in men, 4.51 (1.29-15.7) in women and 1.31 (0.70-2.47) in the total subjects; the ORs and 95% CIs for IS per 5 µmol/L increase in homocysteine were 1.11 (0.99-1.22), 1.25 (1.03-1.58) and 1.15 (1.01-1.28) in men, women and total subjects, respectively. We observed significant associations in crude model, Model 1 and Model 2 in women for the comparison of tHcy ≥ 15 µmol/L versus < 15 µmol/L. Interaction analysis showed that the association of tHcy with IS was significant in women (p-interaction = 0.04). CONCLUSION: This matched case-control study indicates that tHcy may increase the susceptibility to IS in essential hypertension subjects, especially in women. Further large prospective cohort studies are needed to confirm our findings.

A ubiquitin-like domain recruits an oligomeric chaperone to a retrotranslocation complex in endoplasmic reticulum-associated degradation.

The Bag6-Ubl4A-Trc35 complex is a multifunctional chaperone that regulates various cellular processes. The diverse functions of Bag6 are supported by its ubiquitous localization to the cytoplasm, the nucleus, and membranes of the endoplasmic reticulum (ER) in cells. In ER-associated degradation (ERAD) pathways, Bag6 can interact with the membrane-associated ubiquitin ligase gp78 via its ubiquitin-like (UBL) domain, but the relative low affinity of this interaction does not reconcile with the fact that a fraction of Bag6 is tightly bound to the membranes. Here, we demonstrate that the UBL domain of Bag6 is required for interaction with the ER membranes. We find that in addition to gp78, the Bag6 UBL domain also binds a UBL-binding motif in UbxD8, an essential component of the gp78 ubiquitinating machinery. Importantly, Bag6 contains a proline-rich (PR) domain termed PDP (Proline rich-DUF3587-Proline rich) that forms homo-oligomer, allowing the UBL domain to form multivalent interactions with gp78 and UbxD8, which are essential for recruitment of Bag6 to the ER membrane. Furthermore, the PR domain comprises largely intrinsically disordered segments, which are sufficient for interaction with an unfolded substrate. We propose that simultaneous association with multiple ERAD factors helps to anchor a disordered chaperone oligomer to the site of retrotranslocation to prevent protein aggregation in ERAD.

The Cardiovascular Benefits of Glucagon-Like Peptide-1 Receptor Agonists as Novel Diabetes Drugs Are Mediated via the Suppression of miR-203a-3p and miR-429 Expression.

Coronary artery disease (CAD) is associated with a high fatality rate and a heavy global health care burden. Glucagon-like peptide-1 (GLP-1) exerts positive cardiovascular effects, although the molecular mechanisms are unclear. Therefore, this study aimed to verify whether the cardioprotective effects of GLP-1 are mediated through the regulation of micro-RNA (miRNA) expression. Follow-up assessments were conducted for 116 patients with type 2 diabetes mellitus (T2DM) alone (controls) and 123 patients with both T2DM and CAD. After matching, each group comprised 63 patients, and age, body mass index, and serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), triglycerides (TG), and hemoglobin A1C (HbA1c) were compared. Subsequently, the expression profiles of four circulating miRNAs (miR-203a-3p, miR-429, miR-205-5p, and miR-203b-5p) were assessed via quantitative reverse transcription real-time polymerase chain reaction in the 63 patients with diabetes and CAD between 6 months (baseline) and 12 months after the initiation of GLP-1 receptor (GLP-1R) therapy. As expected, the metabolic factors were significantly improved after 6 months of treatment with GLP-1R compared with pre-treatment values, and the expression levels of two of the miRNAs (miR-203a-3p and miR-429) decreased from baseline levels in those with diabetes and CAD. The results suggest that the cardiovascular benefits induced by GLP-1R are mediated via suppressed expression of two miRNAs: miR-203a-3p and miR-429.

mtROS-mediated mitophagy is involved in aflatoxin-B1 induced liver injury in ducks.

Aflatoxin B1 (AFB1) is highly toxic to the liver and can cause excessive production of mitochondrial reactive oxygen species (mtROS) in hepatocytes, leading to oxidative stress, inflammation, fibrosis, cirrhosis, and even liver cancer. The overproduction of mtROS can induce mitophagy, but whether mtROS and mitophagy are involved in the liver injury induced by AFB1 in ducks remains unclear. In this study, we first demonstrated that overproduction of mtROS and mitophagy occurred during liver injury induced by AFB1 exposure in ducks. Then, by inhibiting mtROS and mitophagy, we found that the damage caused by AFB1 in ducks was significantly alleviated, and the overproduction of mtROS induced by AFB1 exposure could mediate the occurrence of mitophagy. These results suggested that mtROS-mediated mitophagy is involved in AFB1-induced duck liver injury, and they may be the prevention and treatment targets of AFB1 hepatotoxicity.

Single [0001]-oriented zinc metal anode enables sustainable zinc batteries.

The optimization of crystalline orientation of a Zn metal substrate to expose more Zn(0002) planes has been recognized as an effective strategy in pursuit of highly reversible Zn metal anodes. However, the lattice mismatch between substrate and overgrowth crystals has hampered the epitaxial sustainability of Zn metal. Herein, we discover that the presence of crystal grains deviating from [0001] orientation within a Zn(0002) metal anode leads to the failure of epitaxial mechanism. The electrodeposited [0001]-uniaxial oriented Zn metal anodes with a single (0002) texture fundamentally eliminate the lattice mismatch and achieve ultra-sustainable homoepitaxial growth. Using high-angle angular dark-filed scanning transmission electron microscopy, we elucidate the homoepitaxial growth of the deposited Zn following the "~ABABAB~" arrangement on the Zn(0002) metal from an atomic-level perspective. Such consistently epitaxial behavior of Zn metal retards dendrite formation and enables improved cycling, even in Zn||NH4V4O10 pouch cells, with a high capacity of 220 mAh g-1 for over 450 cycles. The insights gained from this work on the [0001]-oriented Zn metal anode and its persistently homoepitaxial mechanism pave the way for other metal electrodes with high reversibility.

Correlation Between Thyroid-Related Hormones and Diabetic Retinopathy in Type 2 Diabetes Mellitus Patients with Normal Thyroid Function: A Retrospective Study.

Purpose: To investigate the correlation between thyroid-related hormones and diabetic retinopathy (DR) in euthyroid patients with type 2 diabetes mellitus (T2DM). Patients and Methods: Patients with T2DM admitted to our hospital between January 2023 and June 2023 were retrospectively analyzed. The patients were divided into DR and non-diabetic retinopathy (NDR) groups according to whether DR occurred. Thyroid function-related hormones (TSH, FT3, and FT4), blood glucose indices (FBG and HbA1c), and blood lipid indices (HDL-C, LDL-C, TC, and TG) of the two groups were analyzed by univariate and multivariate logistic regression to explore the risk factors for DR. Pearson correlation analysis and multiple stepwise regression analysis were used to investigate the correlation of TSH or FT3 with FBG, HbA1c, and TG in DR patients. Results: Of the 286 patients with T2DM included in this study, 101 (35.31%) developed DR and 185 (64.69%) did not. High TG, FBG, HbA1c, and TSH and low FT3 levels were independent risk factors for DR in T2DM patients. TSH positively correlated with TG, whereas FT3 negatively correlated with TG and HbA1c in T2DM patients with DR. Conclusion: Higher TSH and lower FT3 in T2DM patients with normal thyroid function may affect glucose and lipid metabolism, thereby increasing the risk of DR.

USP20 deubiquitinates and stabilizes the reticulophagy receptor RETREG1/FAM134B to drive reticulophagy.

The endoplasmic reticulum (ER) serves as a hub for various cellular processes, and maintaining ER homeostasis is essential for cell function. Reticulophagy is a selective process that removes impaired ER subdomains through autophagy-mediatedlysosomal degradation. While the involvement of ubiquitination in autophagy regulation is well-established, its role in reticulophagy remains unclear. In this study, we screened deubiquitinating enzymes (DUBs) involved in reticulophagy and identified USP20 (ubiquitin specific peptidase 20) as a key regulator of reticulophagy under starvation conditions. USP20 specifically cleaves K48- and K63-linked ubiquitin chains on the reticulophagy receptor RETREG1/FAM134B (reticulophagy regulator 1), thereby stabilizing the substrate and promoting reticulophagy. Remarkably, despite lacking a transmembrane domain, USP20 is recruited to the ER through its interaction with VAPs (VAMP associated proteins). VAPs facilitate the recruitment of early autophagy proteins, including WIPI2 (WD repeat domain, phosphoinositide interacting 2), to specific ER subdomains, where USP20 and RETREG1 are enriched. The recruitment of WIPI2 and other proteins in this process plays a crucial role in facilitating RETREG1-mediated reticulophagy in response to nutrient deprivation. These findings highlight the critical role of USP20 in maintaining ER homeostasis by deubiquitinating and stabilizing RETREG1 at distinct ER subdomains, where USP20 further recruits VAPs and promotes efficient reticulophagy.Abbreviations: ACTB actin beta; ADRB2 adrenoceptor beta 2; AMFR/gp78 autocrine motility factor receptor; ATG autophagy related; ATL3 atlastin GTPase 3; BafA1 bafilomycin A1; BECN1 beclin 1; CALCOCO1 calcium binding and coiled-coil domain 1; CCPG1 cell cycle progression 1; DAPI 4',6-diamidino-2-phenylindole; DTT dithiothreitol; DUB deubiquitinating enzyme; EBSS Earle's Balanced Salt Solution; FFAT two phenylalanines (FF) in an acidic tract; GABARAP GABA type A receptor-associated protein; GFP green fluorescent protein; HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase; IL1B interleukin 1 beta; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; PIK3C3/Vps34 phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200 RB1 inducible coiled-coil 1; RETREG1/FAM134B reticulophagy regulator 1; RFP red fluorescent protein; RHD reticulon homology domain; RIPK1 receptor interacting serine/threonine kinase 1; RTN3L reticulon 3 long isoform; SEC61B SEC61 translocon subunit beta; SEC62 SEC62 homolog, preprotein translocation factor; SIM super-resolution structured illumination microscopy; SNAI2 snail family transcriptional repressor 2; SQSTM1/p62 sequestosome 1; STING1/MITA stimulator of interferon response cGAMP interactor 1; STX17 syntaxin 17; TEX264 testis expressed 264, ER-phagy receptor; TNF tumor necrosis factor; UB ubiquitin; ULK1 unc-51 like autophagy activating kinase 1; USP20 ubiquitin specific peptidase 20; USP33 ubiquitin specific peptidase 33; VAMP8 vesicle associated membrane protein 8; VAPs VAMP associated proteins; VMP1 vacuole membrane protein 1; WIPI2 WD repeat domain, phosphoinositide interacting 2; ZFYVE1/DFCP1 zinc finger FYVE-type containing 1.

Peste des petits ruminants virus infection induces endoplasmic reticulum stress and apoptosis via IRE1-XBP1 and IRE1-JNK signaling pathways.

BACKGROUND: Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The activation of UPR signaling pathways and their impact on apoptosis and virus replication remains controversial. OBJECTIVES: To investigate the role of PPRV-induced ER stress and the IRE1-XBP1 and IRE1-JNK pathways and their impact on apoptosis and virus replication. METHODS: The cell viability and virus replication were assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, immunofluorescence assay, and Western blot. The expression of ER stress biomarker GRP78, IRE1, and its downstream molecules, PPRV-N protein, and apoptosis-related proteins was detected by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. 4-Phenylbutyric acid (4-PBA) and STF-083010 were respectively used to inhibit ER stress and IRE1 signaling pathway. RESULTS: The expression of GRP78, IRE1α, p-IRE1α, XBP1s, JNK, p-JNK, caspase-3, caspase-9, Bax and PPRV-N were significantly up-regulated in PPRV-infected cells, the expression of Bcl-2 was significantly down-regulated. Due to 4-PBA treatment, the expression of GRP78, p-IRE1α, XBP1s, p-JNK, caspase-3, caspase-9, Bax, and PPRV-N were significantly down-regulated, the expression of Bcl-2 was significantly up-regulated. Moreover, in PPRV-infected cells, the expression of p-IRE1α, p-JNK, Bax, and PPRV-N was significantly decreased, and the expression of Bcl-2 was increased in the presence of STF-083010. CONCLUSIONS: PPRV infection induces ER stress and IRE1 activation, resulting in apoptosis and enhancement of virus replication through IRE1-XBP1s and IRE1-JNK pathways.