UBA2 as a Prognostic Biomarker and Potential Therapeutic Target in Glioma.

BACKGROUND: Gliomas are characterized by aggressive behavior, leading to severe disability and high mortality. Ubiquitin-like modifier activating enzyme 2 (UBA2) is a subunit of the E1-activating enzyme involved in the SUMOylation (SUMO, small ubiquitin-related modifier) of numerous proteins. Although the abnormality of UBA2 is linked to the progression of various tumor types, the role of UBA2 in glioma is still unknown. METHODS: A bioinformatic analysis using several public databases was conducted to examine the expression level, clinicopathological correlations, and prognostic significance of UBA2 in glioma. The correlation between UBA2 expression and drug sensitivity in cancers was also explored. Multiple cellular experiments were conducted to validate the role of UBA2 in glioma. RESULTS: Analysis of multiple databases and cellular experiments revealed that UBA2 was overexpressed in glioma tissues and cell lines, respectively. UBA2 expression in gliomas correlated with World Health Organization (WHO) grade, IDH gene status, 1p19q deletion, histological type, and immune cell infiltration in glioma. UBA2 expression in carcinomas also correlated with drug sensitivity. Kaplan-Meier analysis revealed that high expression of UBA2 predicted poorer survival in glioma patients. A nomogram model containing UBA2 expression was constructed for clinical practice. Knockdown of UBA2 was observed to suppress glioma cell progression and sensitize glioma cells to irradiation in vitro. CONCLUSION: Overall, this research showed that UBA2 might be involved not only in the development of glioma but also in the regulation of immunity, drug sensitivity, and radiosensitivity. Therefore, UBA2 may be a potential target for therapy and a candidate biomarker for glioma diagnosis and prognosis.

Cortical Proteins Associated With Cognitive Resilience in Community-Dwelling Older Persons.

Importance: Identifying genes and proteins for cognitive resilience (ie, targets that may be associated with slowing or preventing cognitive decline regardless of the presence, number, or combination of common neuropathologic conditions) provides a complementary approach to developing novel therapeutics for the treatment and prevention of Alzheimer disease and related dementias. Objective: To identify proteins associated with cognitive resilience via a proteome-wide association study of the human dorsolateral prefrontal cortex. Design, Setting, and Participants: This study used data from 391 community-dwelling older persons who participated in the Religious Orders Study and the Rush Memory and Aging Project. The Religious Orders Study began enrollment January 1, 1994, and the Rush Memory and Aging Project began enrollment September 1, 1997, and data were collected and analyzed through October 23, 2019. Exposures: Participants had undergone annual detailed clinical examinations, postmortem evaluations, and tandem mass tag proteomics analyses. Main Outcomes and Measures: The outcome of cognitive resilience was defined as a longitudinal change in cognition over time after controlling for common age-related neuropathologic indices, including Alzheimer disease, Lewy bodies, transactive response DNA-binding protein 43, hippocampal sclerosis, infarcts, and vessel diseases. More than 8000 high abundance proteins were quantified from frozen dorsolateral prefrontal cortex tissue using tandem mass tag and liquid chromatography-mass spectrometry. Results: There were 391 participants (273 women); their mean (SD) age was 79.7 (6.7) years at baseline and 89.2 (6.5) years at death. Eight cortical proteins were identified in association with cognitive resilience: a higher level of NRN1 (estimate, 0.140; SE, 0.024; P = 7.35 × 10-9), ACTN4 (estimate, 0.321; SE, 0.065; P = 9.94 × 10-7), EPHX4 (estimate, 0.198; SE, 0.042; P = 2.13 × 10-6), RPH3A (estimate, 0.148; SE, 0.031; P = 2.58 × 10-6), SGTB (estimate, 0.211; SE, 0.045; P = 3.28 × 10-6), CPLX1 (estimate, 0.136; SE, 0.029; P = 4.06 × 10-6), and SH3GL1 (estimate, 0.179; SE, 0.039; P = 4.21 × 10-6) and a lower level of UBA1 (estimate, -0.366; SE, 0.076; P = 1.43 × 10-6) were associated with greater resilience. Conclusions and Relevance: These protein signals may represent novel targets for the maintenance of cognition in old age.

MLN4924 suppresses neddylation and induces cell cycle arrest, senescence, and apoptosis in human osteosarcoma.

Neddylation is a post-translational protein modification process associated with carcinogenesis and cancer development. MLN4924, a pharmaceutical neddylation inhibitor, induces potent anti-cancer effects in multiple types of cancers. In this study, we investigated the effects of MLN4924 on human osteosarcoma (OS). Levels of both NEDD8 activating enzyme E1 (NAE1) and ubiquitin-conjugating enzyme E2M (Ube2M), two critical components of the neddylation pathway, were much higher in OS tissues and cells than in normal osseous tissues and cells. MLN4924 treatment led to DNA damage, reduced cell viability, senescence and apoptosis in OS cells. Moreover, MLN4924 inhibited OS xenograft tumor growth in mice. Mechanistically, MLN4924 blocked the neddylation of cullins and induced accumulation of several tumor-suppressive substrates of Cullin-RING E3 ubiquitin ligases (CRLs), including CDT1, Wee1, p21, p27, Noxa, and p16. These results suggest clinical studies investigating the utility of MLN4924 for the treatment of OS are warranted.

[Hashimoto's encephalopathy presenting with vertigo and muscle weakness in a male pediatric patient].

Hashimoto's encephalopathy is an anti-thyroid antibody-positive autoimmune encephalopathy. We herein report the case of a 13-year-old male patient with subacute vertigo, muscle weakness in the extremities and gait disturbance who was diagnosed with Hashimoto's encephalopathy. He showed no severe impairment of consciousness and no seizures, and there were no abnormalities on the brain MRI. However, epileptic spike and wave complexes were observed on an electroencephalogram, and a decline in blood flow was diffusely observed on brain SPECT (single photon emission computed tomography). His thyroid function was normal, but he was positive for anti-thyroid antibodies, such as anti-TPO (thyroid peroxidase) antibodies. He was also positive for serum anti-NAE (NH2-terminal alpha-enolase) antibodies. Systemic corticosteroid therapy and high-dose intravenous immunoglobulin therapy were effective, greatly improving his quality of life.

Autophagy in the Degenerating Human Intervertebral Disc: In Vivo Molecular and Morphological Evidence, and Induction of Autophagy in Cultured Annulus Cells Exposed to Proinflammatory Cytokines-Implications for Disc Degeneration.

STUDY DESIGN: Autophagy-related gene expression and ultrastructural features of autophagy were studied in human discs. OBJECTIVE: To obtain molecular/morphological data on autophagy in human disc degeneration and cultured human annulus cells exposed to proinflammatory cytokines. SUMMARY OF BACKGROUND DATA: Autophagy is an important process by which cytoplasm and organelles are degraded; this adaptive response to sublethal stresses (such as nutrient deprivation present in disc degeneration) supplies needed metabolites. Little is known about autophagic processes during disc degeneration. METHODS: Human disc specimens were obtained after institutional review board approval. Annulus mRNA was analyzed to determine autophagy-related gene expression levels. Immunolocalization and ultrastructural studies for p62, ATG3, ATG4B, ATG4C, ATG7, L3A, ULK-2, and beclin were conducted. In vitro experiments used IL-1ß- or TNF-α-treated human annulus cells to test for autophagy-related gene expression. RESULTS: More degenerated versus healthier discs showed significantly greater upregulation of well-recognized autophagy-related genes (P ≤ 0.028): beclin 1 (upregulated 1.6-fold); ATG8 (LC3) (upregulated 2.0-fold); ATG12 (upregulated 4.0-fold); presenilin 1 (upregulated 1.6-fold); cathepsin B (upregulated 4.5-fold). p62 was localized, and ultrastructure showed autophagic vacuolization and autophagosomes with complex, redundant whorls of membrane-derived material. In vitro, proinflammatory cytokines significantly upregulated autophagy-related genes (P ≤ 0.04): DRAM1 (6.24-fold); p62 (4.98-fold); PIM-2 oncogene, a positive regulator of autophagy (3-fold); WIPI49 (linked to starvation-induced autophagy) (upregulated 2.3-fold). CONCLUSION: Data provide initial molecular and morphological evidence for the presence of autophagy in the degenerating human annulus. In vivo gene analyses showed greater autophagy-related gene expression in more degenerated than healthier discs. In vitro data suggested a mechanism implicating a role of TNF-α and IL-1ß in disc autophagy. Findings suggest the importance of future work to investigate the relationship of autophagy to apoptosis, cell death, cell senescence, and mitochondrial dysfunction in the aging and degenerating disc. LEVEL OF EVIDENCE: N/A.

Increased expression of autophagy-related proteins in keratocystic odontogenic tumours: its possible association with growth potential.

The aim of this study was to evaluate the activation status of autophagy in keratocystic odontogenic tumours (KCOT), and to investigate its possible association with growth potential. We detected the expression of some key autophagy-related proteins in clinical samples of KCOT and radicular cysts and compared then by real-time quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, respectively. The correlation between the autophagy-related proteins tested, and with cell antiapoptotic (Bcl-2) or proliferative (Ki-67) markers in KCOT was explored using Spearman's rank correlation, followed by cluster analysis. The results showed that both the expression of mRNA and the immunoreactivity of the autophagy-related proteins tested were considerably increased in samples of KCOT compared with those in samples of radicular cysts. The correlation analyses showed that the immunostains of autophagy-related proteins in samples of KCOT correlated closely with each other. The immunostains of these autophagy-related proteins also correlated closely with the immunostains of Bcl-2 and Ki-67 in KCOT. More importantly, double-labelling immunofluorescence analyses also showed that the distribution of autophagic and proliferative markers was partially synchronous in samples from KCOT. We have, to our knowledge for the first time, implicated the activation of autophagy in KCOT, and showed its possible association with growth potential.

Immunohistochemical assessment of ATG7, LC3, and p62 in ameloblastomas.

BACKGROUND: To investigate the roles of autophagy in tumorigenesis, cytodifferentiation, and prognosis of odontogenic tumors, we analyzed the immunohistochemical expression of ATG7, LC3, and p62 in odontogenic tissues. METHODS: Tissue specimens of nine dental follicles and 69 ameloblastomas were immunohistochemically examined with antibodies against ATG7, LC3, and p62. RESULTS: Immunohistochemical reactivity for ATG7, LC3, and p62 was detected in many odontogenic epithelial cells and several endothelial cells and fibroblasts in dental follicles and ameloblastomas. ATG7 reactivity in ameloblatomas was significantly higher than that in dental follicles. Expression of ATG7, LC3, and p62 was found markedly in neoplastic cells near the basement membrane rather than central polyhedral cells in ameloblastomas. Reactivity for these molecules was significantly higher in unicystic ameloblastomas than in solid ameloblastomas. Granular cells in granular cell ameloblastomas showed obvious reactivity for the autophagy- related molecules, and LC3 reactivity in granular cell ameloblastomas was significantly higher than in other ameloblastoma variations. Recurrent ameloblastomas showed significantly lower reactivity of LC3 and p62 than primary ameloblastomas. CONCLUSIONS: Expression of ATG7, LC3, and p62 in dental follicles and ameloblastomas suggests that autophagy regulation might be affected by microenvironment alterations during tumorigenesis. The molecular machinery for autophagy is possibly involved in tissue architecture, neoplastic cell differentiation, and prognosis of the benign epithelial odontogenic tumor.

Impaired autophagy: A mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.

UNLABELLED: Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy-related protein 7 (Atg7), Beclin-1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid-free and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. CONCLUSION: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy, and this subsequently leads to MPT-dependent hepatocyte death after A/R.

Involvement of ubiquitination and sumoylation in bladder lesions induced by persistent long-term low dose ionizing radiation in humans.

PURPOSE: We determined whether ubiquitination and sumoylation processes are up-regulated in bladder urothelium by chronic, long-term, persistent low doses of ionizing radiation in male patients with benign prostate hyperplasia and females with chronic cystitis living more than 19 years in 137Cs contaminated areas after the Chernobyl accident in Ukraine. MATERIALS AND METHODS: Bladder urothelial biopsies from 45 patients were subjected to histopathological and immunohistochemical study of Ub, SUMO1, SUMO E2 conjugating enzyme Ubc9, and the cell cycle inhibitors p53 and p27(Kip1). RESULTS: Of 25 group 1 patients from radio contaminated areas chronic proliferative atypical cystitis (Chernobyl cystitis), featuring multiple foci of dysplasia, and carcinoma in situ were observed in 23 (92%) and 19 (76%), respectively, in addition to 1 small pTa grade 1 urothelial carcinoma. Chronic cystitis with areas of dysplasia and urothelial hyperplasia were detected in 2 (10%) and 3 (15%), respectively of the 20 patients in control group 2 from clean (without radio contamination) areas of Ukraine. Greatly increased levels of Ub, SUMO1, Ubc9 and p53 as well as decreased levels of p27(Kip1) were evident in patients in group 1 compared to those in group 2 (all p <0.001). CONCLUSIONS: These findings support the hypothesis that up-regulated ubiquitination and sumoylation processes might be an adaptive response to unscheduled proteolysis of aberrant p53 and p27(Kip1) cell cycle regulators occurring with long-term low dose rate ionizing radiation exposure with a possible contribution to urothelial carcinogenesis.

Studies of the ubiquitin proteasome system.

A concept that has arisen over the last decade is that proteins can, in general, be covalently modified by polypeptides, resulting in alterations in their fate and function. The first-identified and most well studied of these modifying polypeptides is ubiquitin. Although targeting for proteasomal degradation is the best studied outcome of ubiquitylation, we now understand that modification of proteins with ubiquitin has numerous other cellular roles that alter protein function and that are unrelated to proteasomal degradation. Ubiquitylation is a complex process that is regulated at the level of both addition and removal of ubiquitin from target proteins. This unit includes a number of different basic protocols that will facilitate the study of components of the ubiquitin system and substrate ubiquitylation both in vitro and in cells. Because another protein modifier, NEDD8, itself regulates aspects of the ubiquitin system, basic protocols on neddylation are also included in this unit.

Microarray analyses uncover UBE1L as a candidate target gene for lung cancer chemoprevention.

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.

Preferred in vivo ubiquitination sites.

MOTIVATION: The conjugation of ubiquitin to target molecules involves several enzymatic steps. Little is known about the specificity of ubiquitination. How E3 ligases select their substrate and which lysines are targeted for ubiquitin conjugation is largely an enigma. The object of this study is to identify preferred ubiquitination sites. Genetic approaches to study this question have proven difficult, because of the redundancy of ligases and the lack of strictly required motifs. However, a better understanding of acceptor site selection could help to predict ubiquitination sites and clarify yet unsolved structure-function relationships of the transfer reaction. RESULTS: In an effort to define preferences for ubiquitination, we systematically analyzed structure and sequence of 135 known ubiquitination sites in 95 proteins in Saccharomyces cerevisiae. The results show clear structural preferences for ubiquitin ligation to target proteins, and compartment-specific amino acid patterns in close proximity to the modified side chain. SUPPLEMENTARY INFORMATION: http://www.people.fas.harvard.edu/~catic.