Inhibition of UBA52 induces autophagy via EMC6 to suppress hepatocellular carcinoma tumorigenesis and progression.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52) has a role in the occurrence and development of tumours. However, the mechanism by which UBA52 regulates hepatocellular carcinoma (HCC) tumorigenesis and progression remains poorly understood. By using the Cell Counting Kit (CCK-8), colony formation, wound healing and Transwell assays, we assessed the effects of UBA52 knockdown and overexpression on the proliferation and migration of HCC cells in vitro. By establishing subcutaneous and metastatic tumour models in nude mice, we evaluated the effects of UBA52 on HCC cell proliferation and migration in vivo. Through bioinformatic analysis of data from the Gene Expression Profiling Interactive Analysis (GEPIA) and The Cancer Genome Atlas (TCGA) databases, we discovered that UBA52 is associated with autophagy. In addition, we discovered that HCC tissues with high UBA52 expression had a poor prognosis in patients. Moreover, knockdown of UBA52 reduced HCC cell growth and metastasis both in vitro and in vivo. Mechanistically, knockdown of UBA52 induced autophagy through EMC6 in HCC cells. These findings suggest that UBA52 promoted the proliferation and migration of HCC cells through autophagy regulation via EMC6 and imply that UBA52 may be a viable novel treatment target for HCC patients.

Hydralazine represses Fpn ubiquitination to rescue injured neurons via competitive binding to UBA52.

A major impedance to neuronal regeneration after peripheral nerve injury (PNI) is the activation of various programmed cell death mechanisms in the dorsal root ganglion. Ferroptosis is a form of programmed cell death distinguished by imbalance in iron and thiol metabolism, leading to lethal lipid peroxidation. However, the molecular mechanisms of ferroptosis in the context of PNI and nerve regeneration remain unclear. Ferroportin (Fpn), the only known mammalian nonheme iron export protein, plays a pivotal part in inhibiting ferroptosis by maintaining intracellular iron homeostasis. Here, we explored in vitro and in vivo the involvement of Fpn in neuronal ferroptosis. We first delineated that reactive oxygen species at the injury site induces neuronal ferroptosis by increasing intracellular iron via accelerated UBA52-driven ubiquitination and degradation of Fpn, and stimulation of lipid peroxidation. Early administration of the potent arterial vasodilator, hydralazine (HYD), decreases the ubiquitination of Fpn after PNI by binding to UBA52, leading to suppression of neuronal cell death and significant acceleration of axon regeneration and motor function recovery. HYD targeting of ferroptosis is a promising strategy for clinical management of PNI.

Omics Analyses in a Neural Stem Cell Model of Familial Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting millions of people worldwide. Despite considerable efforts, the underlying pathological mechanisms remain elusive, and yet, no treatment has been developed to efficiently reverse or modify disease progression. Thus, new experimental models are required to provide insights into the pathology of PD. Small-molecule neural precursor cells (smNPCs) are ideal for the study of neurodegenerative disorders due to their neural identity and stem cell properties. Cytoplasmic aggregates of α-synuclein (αSyn) are considered a hallmark of PD and a point mutation in the gene encoding p.A53T is responsible for a familial PD form with earlier and robust symptom onset. In order to study the cellular pathology of PD, we genetically modified smNPCs to inducibly overexpress EYFP-SNCA A53T. This cellular model was biochemically characterized, while dysregulated biological pathways and key regulators of PD pathology were identified by computational analyses. Our study indicates three novel genes, UBA52, PIP5K1A, and RPS2, which may mediate PD cellular pathology.

UBA52 Attunes VDAC1-Mediated Mitochondrial Dysfunction and Dopaminergic Neuronal Death.

Mitochondrial homeostasis regulates energy metabolism, calcium buffering, cell function, and apoptosis. The present study has been conducted to investigate the implications of the ubiquitin-encoding gene UBA52 in mitochondrial physiology. Transient expression of Myc-UBA52 in neurons significantly inhibited the rotenone-induced increase in reactive oxygen species generation, nitrite level, and depleted glutathione level. Mass spectrometric and coimmunoprecipitation data suggested the profound interaction of UBA52 with mitochondrial outer membrane channel protein, VDAC1 in both the wild-type and Myc-α-synuclein overexpressed neuronal cells and in the Parkinson's disease (PD)-specific substantia nigra and striatal region of the rat brain. In vitro ubiquitylation assay revealed that UBA52 participates in the ubiquitylation of VDAC1 through E3 ligase CHIP. Myc-UBA52 overexpression in neurons further improved the mitochondrial functionality and cell viability by preventing the alteration in mitochondrial membrane potential, mitochondrial complex I activity, and translocation of cytochrome c and p-Nrf2 along with the effect on intracellular calcium uptake, thus collectively inhibiting the opening of mitochondrial permeability transition pore. Additionally, Myc-UBA52 expression in neuronal cells offered protection against apoptotic and autophagic cell death. Altogether, our findings delineate a functional association between UBA52 and mitochondrial homeostasis, providing new insights into the deterrence of dopaminergic cell death during acute PD pathogenesis.

UBA52 Is Crucial in HSP90 Ubiquitylation and Neurodegenerative Signaling during Early Phase of Parkinson's Disease.

Protein aggregation is one of the major pathological events in age-related Parkinson's disease (PD) pathology, predominantly regulated by the ubiquitin-proteasome system (UPS). UPS essentially requires core component ubiquitin; however, its role in PD pathology is obscure. This study aimed to investigate the role of ubiquitin-encoding genes in sporadic PD pathology. Both cellular and rat models of PD as well as SNCA C57BL/6J-Tg (Th-SNCA*A30P*A53T)39 Eric/J transgenic mice showed a decreased abundance of UBA52 in conjunction with significant downregulation of tyrosine hydroxylase (TH) and neuronal death. In silico predictions, mass spectrometric analysis, and co-immunoprecipitation findings suggested the protein-protein interaction of UBA52 with α-synuclein, HSP90 and E3-ubiquitin ligase CHIP, and its co-localization with α-synuclein in the mitochondrion. Next, in vitro ubiquitylation assay indicated an imperative requirement of the lysine-63 residue of UBA52 in CHIP-mediated HSP90 ubiquitylation. Myc-UBA52 expressed neurons inhibited alteration in PD-specific markers such as α-synuclein and TH protein along with increased proteasome activity in diseased conditions. Furthermore, Myc-UBA52 expression inhibited the altered protein abundance of HSP90 and its various client proteins, HSP75 (homolog of HSP90 in mitochondrion) and ER stress-related markers during early PD. Taken together, the data highlights the critical role of UBA52 in HSP90 ubiquitylation in parallel to its potential contribution to the modulation of various disease-related neurodegenerative signaling targets during the early phase of PD pathology.

Clinical significance of UbA52 level in the urine of patients with type 2 diabetes mellitus and diabetic kidney disease / Importancia clínica del nivel de UbA52 en la orina de pacientes con diabetes mellitus tipo 2 y enfermedad renal diabética

Background: Ubiquitin-52 amino acid fusion protein (UbA52) is an important factor in the pathogenesis of diabetic kidney disease (DKD) and has been suggested a potential marker in the disease. However, whether upregulation of UbA52 marks early kidney injury in T2DM mellitus (T2DM) patients remains unclear. In this study, we examine the diagnostic value of UbA52 as a biomarker in predicting early diabetic kidney disease (DKD) in T2DM patients. Methods: We used two-step ELISA to test UbA52 level in urine of 3 defined patient groups. Samples from T2DM patients without albuminuria or diabetic retinopathy (DM-WNP; n=30), T2DM patients with albuminuria and diabetic retinopathy, excluding other renal diseases clinically (DM-NP; n=30) and healthy controls (n=30) were analyzed. Spearman's correlation analysis and multiple linear regression model were used to analyze the correlation of urinary UbA52 level with laboratory results regarding kidney function. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of UbA52 in predicting T2DM and early DKD. Results: Urinary UbA52 level in DM-NP group was 1.75 times and 2.71 times higher than in DN-WNP (p=0.004) and normal control group (p<0.001), respectively. The level of urinary UbA52 correlated significantly with serum creatinine (r=0.468, p<0.001), GFR (r=−0.300, p=0.004) and proteinuria (r=0.484, p<0.001). Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level (β=0.833, p<0.001). The area under the ROC of urinary UbA52 in diagnosing T2DM and DKD was 0.751 and 0.755, respectively. (AU)

Antecedentes: La proteína de fusión de aminoácidos ubiquitina-52 (UbA52) es un factor importante en la patogénesis de la enfermedad renal diabética (ERD), y se ha sugerido como marcador potencial en la enfermedad. Sin embargo, aún no está claro si la regulación al alza de UbA52 indica una lesión renal temprana en pacientes con diabetes mellitus de tipo 2 (DMT2). En este estudio, analizamos el valor diagnóstico de UbA52 como biomarcador para predecir la ERD temprana en pacientes con DMT2. Métodos: Utilizamos un ELISA de 2 pasos para analizar el nivel de UbA52 en la orina de 3 grupos de pacientes definidos. Se analizaron muestras de pacientes con DMT2 sin albuminuria o retinopatía diabética (DM-WNP; n=30), pacientes con DMT2 y con albuminuria y retinopatía diabética, excluyendo clínicamente otras enfermedades renales (DM-NP; n=30) y controles sanos (n=30). Se utilizó el análisis de correlación de Spearman y el modelo de regresión lineal múltiple para analizar la correlación del nivel de UbA52 en orina con los resultados de laboratorio relativos a la función renal. Se utilizó la curva de características operativas del receptor (ROC) para evaluar el valor diagnóstico de UbA52 en la predicción de la DMT2 y de la ERD temprana. Resultados: El nivel de UbA52 en orina en el grupo DM-NP fue 1,75 y 2,71 veces mayor que en el grupo DN-WNP (p=0,004), y en el grupo de control normal (p<0,001), respectivamente. El nivel de UbA52 en orina se correlacionó significativamente con la creatinina sérica (r=0,468; p<0,001), la TFG (r=−0,300; p=0,004) y la proteinuria (r=0,484; p<0,001). El análisis de regresión lineal múltiple mostró que el nivel de proteinuria se asociaba de forma independiente al nivel de UbA52 en orina (β=0,833; p<0,001). El área bajo las ROC de UbA52 en orina en el diagnóstico de la DMT2 y de la ERD fue de 0,751 y 0,755, respectivamente. (AU)

Clinical significance of UbA52 level in the urine of patients with type 2 diabetes mellitus and diabetic kidney disease.

BACKGROUND: Ubiquitin-52 amino acid fusion protein (UbA52) is an important factor in the pathogenesis of diabetic kidney disease (DKD) and has been suggested a potential marker in the disease. However, whether upregulation of UbA52 marks early kidney injury in T2DM mellitus (T2DM) patients remains unclear. In this study, we examine the diagnostic value of UbA52 as a biomarker in predicting early diabetic kidney disease (DKD) in T2DM patients. METHODS: We used two-step ELISA to test UbA52 level in urine of 3 defined patient groups. Samples from T2DM patients without albuminuria or diabetic retinopathy (DM-WNP; n=30), T2DM patients with albuminuria and diabetic retinopathy, excluding other renal diseases clinically (DM-NP; n=30) and healthy controls (n=30) were analyzed. Spearman's correlation analysis and multiple linear regression model were used to analyze the correlation of urinary UbA52 level with laboratory results regarding kidney function. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of UbA52 in predicting T2DM and early DKD. RESULTS: Urinary UbA52 level in DM-NP group was 1.75 times and 2.71 times higher than in DN-WNP (p=0.004) and normal control group (p<0.001), respectively. The level of urinary UbA52 correlated significantly with serum creatinine (r=0.468, p<0.001), GFR (r=-0.300, p=0.004) and proteinuria (r=0.484, p<0.001). Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level (ß=0.833, p<0.001). The area under the ROC of urinary UbA52 in diagnosing T2DM and DKD was 0.751 and 0.755, respectively. CONCLUSION: The level of urinary UbA52 increased significantly in T2DM patients with DKD. The level of proteinuria is independently associated with urinary UbA52 level. Urinary UbA52 could serve as an early marker in the diagnosis of DKD. CLINICALTRIALS: gov Identifier: NCT02204280.

Circular RNA expression profile in blood according to ischemic stroke etiology.

BACKGROUND: The discovery of novel biomarkers of stroke etiology would be most helpful in management of acute ischemic stroke patients. Recently, circular RNAs (circRNAs) have been proposed as candidate biomarkers of neurological conditions due to its high stability. circRNAs function as sponges, sequestering miRNAs and are involved in most relevant biological functions. Our aim was to identify differentially expressed circRNAs in acute ischemic stroke patients according to stroke etiology. METHODS: A comprehensive expression profile of blood circRNAs was conducted by Arraystar Human circRNA arrays (13,617 probes) on a discovery cohort of 30 stroke patients with different stroke etiologies by TOAST classification. Real-time quantitative PCR (RT-qPCR) was used to validate array results in a cohort of 50 stroke patients. Functional in silico analysis was performed to identify potential interactions with microRNAs (miRNAs) and pathways underlying deregulated circRNAs. RESULTS: A set of 60 circRNAs were found to be upregulated in atherotrombotic versus cardioembolic strokes (fold-change > = 1.5 and p-value ≤ 0.05). Differential expression of hsa_circRNA_102488, originated from UBA52 gene, was replicated in the validation cohort. RNA-binding proteins (RBPs) sites of hsa_circRNA_102488 clustered around AGO2 and FUS proteins. Further functional analysis revealed interactions between deregulated circRNAs and a set of miRNAs involved in stroke-related pathways, such as fatty acid biogenesis or lysine degradation. CONCLUSION: Different stroke subtypes show specific profiles of circRNAs expression. circRNAs may serve as a new source of biomarkers of stroke etiology in acute ischemic stroke patients.

Degradation of CCNB1 mediated by APC11 through UBA52 ubiquitination promotes cell cycle progression and proliferation of non-small cell lung cancer cells.

OBJECTIVE: Mechanism by which CCNB1 regulates the cell cycle progression and its prognostic function in non-squamous non-small cell lung cancer (NSCLC) are necessary to be further elucidated. METHODS: Data retrieved from gene expression omnibus (GEO) and cancer genome atlas (TCGA) combined with clinical data were used. Survival analysis was conducted in public datasets. Proteomics and co-immunoprecipation assays were designed to unravel proteins with interaction with CCNB1. Short hairpin RNA and small interfering RNA as well as overexpressing genes of interest were used. RESULTS: CCNB1 was not implicated in apoptosis, migration and invasion of NSCLC cells. After either knockdown or overexpression of CCNB1, the occurrence of cell cycle arrest in G2/M phase, fewer cloning formation and diminished dimension of xenograft tumors were observed. CCNB1 expression level was clinically associated with several clinicopathological parameters including gender, smoking, T stage and N stage. Survival analysis showed that the higher level of CCNB1, the more dismal outcome in overall survival as well as in disease-free survival. Mechanistically, we confirmed that the role of CCNB1 on cell cycle and cloning formation was dependent on UBA52, which was able to promote degradation of CCNB1; nevertheless, this consequence relied on APC11. Knockdown of APC11 led to cell cycle arrest in G2/M and less cloning formation even in the presence of overexpressed UBA52. Following upregulation of APC11, the protein of CCNB1 degraded with resultant cell cycle progression and more cloning formation. CONCLUSION: Degradation of CCNB1 by APC11 via UBA52 ubiquitylation was critical in cell cycle progression and proliferation of NSCLC cell lines.

LUCAT1 promotes colorectal cancer tumorigenesis by targeting the ribosomal protein L40-MDM2-p53 pathway through binding with UBA52.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in both men and women in the USA. However, the underlying molecular mechanisms that drive CRC tumorigenesis are still not clear. Several studies have reported that long noncoding RNAs (lncRNAs) have important roles in tumor development. Here, we undertook a transcriptome microarray analysis in 6 pairs of CRC tissues and their corresponding adjacent normal tissues. A total of 1705 differentially expressed lncRNAs were detected in CRC tissues at stages I/II and III/IV (fold change greater than or equal to 2 or less than or equal to 0.5). Among them, we found that the lncRNA lung cancer-associated transcript 1 (LUCAT1) was upregulated in CRC tissues and was closely associated with poor overall survival of CRC patients, through analysis of clinical data and The Cancer Genome Atlas. Functional studies indicated that LUCAT1 promoted CRC cell proliferation, apoptosis, migration, and invasion in vitro and in vivo. Furthermore, knockdown of LUCAT1 rendered CRC cells hypersensitive to oxaliplatin treatment. Mechanistically, bioinformatic analysis indicated that low expression of LUCAT1 was associated with the p53 signaling pathway. Chromatin isolation by RNA purification followed by mass spectrometry and RNA immunoprecipitation revealed that LUCAT1 bound with UBA52, which encodes ubiquitin and 60S ribosomal protein L40 (RPL40). We found that RPL40 functions in the ribosomal protein-MDM2-p53 pathway to regulate p53 expression. Taken together, our findings indicate that suppression of LUCAT1 induces CRC cell cycle arrest and apoptosis by binding UBA52 and activating the RPL40-MDM2-p53 pathway. These results implicate LUCAT1 as a potential prognostic biomarker and therapeutic target for CRC.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), a ubiquitin-ribosomal fusion gene, is a major source of ubiquitin protein for covalent modification of proteinaceous substrates recycled by ubiquitin-proteasome system (UPS). Its role in early embryo development has not been studied. Using the CRISPR/Cas9 gene editing tool, the objective of this study was to determine if UBA52 protein is required for mammalian embryogenesis. Matured metaphase II porcine oocytes were injected with CRISPR Cas9+guide RNAs (Uba52 gRNA) or Cas9 without gRNAs as control, followed by in vitro fertilization (IVF) and embryo culture to day 7. Injection of Cas9+gRNAs affected embryo development. On day 4 of embryo culture, the proportion of 2-, 4- and 8-cell stage embryos was significantly different between the Uba52 gRNA and control group (P<0.05), with more 8-cell stage embryos in the control and more 4- and 2-cell stage embryos in the Uba52g RNA group. This delay in the development of Uba52 gRNA embryos occurred at the transition from the 4- to 8-cell stages, around the time of major zygotic genomic activation. The percentage of blastocyst formation on day 7 and the cell number per blastocyst were significantly lower in the Uba52 gRNA group than in the control (P<0.05). Genotyping by PCR and DNA gel electrophoresis analysis showed that 91.8% of embryos that failed to develop to blastocyst had either a monoallelic or a biallelic modification of the Uba52 gene. In comparison, only 24.4% of embryos that reached blastocyst had a monoallelic modification and biallelic editing was not found in any of the blastocysts. Based on immuno-labeling intensity, both UBA52 and proteasome protein levels on days 4 and 7 of culture were significantly lower in the Uba52 gRNA group than in the control (P<0.05), in agreement with UBA52 western blotting-densitometry of day 4 embryos. Morphological examination of blastomere nuclei revealed abnormal nuclear structure in the Uba52 gRNA group, such as reduced size, irregular shapes, nucleus fragmentation and uneven DNA distribution at all stages of embryo development. Nuclear morphology studies of embryos injected with Cas9+gRNAs and co-injected with plasmid DNA encoding nuclear localized GFP further supported these observations. In conclusion, our data indicate that the Uba52 gene is essential in early embryogenesis.

Host Interaction Analysis of PA-N155 and PA-N182 in Chicken Cells Reveals an Essential Role of UBA52 for Replication of H5N1 Avian Influenza Virus.

PA-N155 and PA-N182 proteins were translated from the 11th and 13th start codon AUG of the RNA polymerase acidic protein (PA) mRNA of H5N1 influenza A virus (IAV), which plays an important role in viral replication. Little is known about the interactions between PA-N155 and PA-N182 and the host proteins. This study investigated the interaction landscape of PA-N155 and PA-N182 of H5N1 IAV in chicken cells while their interacting complexes were captured by immunoprecipitation and analyzed by mass spectrometry. A total of 491 (PA-N155) and 302 (PA-N182) interacting proteins were identified. Gene ontology and pathway enrichment analyses showed that proteins of the two interactomes were enriched in RNA processing, viral processing and protein transport, and proteins related to signaling pathways of proteasome, ribosome, and aminoacy1-tRNA biosynthesis were significantly enriched, suggesting their potential roles in H5N1 IAV infection. Comparative analysis of the interactome of PA, PA-N155, and PA-N182 identified UBA52 as a conserved host factor that interacted with all three viral proteins. UBA52 is a fusion protein consisting of ubiquitin at the N terminus and ribosomal protein L40 at the C terminus. Knockdown of UBA52 significantly decreased the titer of H5N1 IAV in chicken cells and was accompanied with attenuated production of proinflammatory cytokines. Our analyses of the influenza-host protein interactomes identified UBA52 as a PA interaction protein for virus replication.