UBQLN2 Research Articles

UBQLN1 links proteostasis and mitochondria function to telomere maintenance in human embryonic stem cells

BackgroundTelomeres consist of repetitive DNA sequences at the chromosome ends to protect chromosomal stability, and primarily maintained by telomerase or occasionally by alternative telomere lengthening of telomeres (ALT) through recombination-based mechanisms. Additional mechanisms that may regulate telomere maintenance remain to be explored. Simultaneous measurement of telomere length and transcriptome in the same human embryonic stem cell (hESC) revealed that mRNA expression levels of UBQLN1 exhibit linear relationship with telomere length.MethodsIn this study, we first generated UBQLN1-deficient hESCs and compared with the wild-type (WT) hESCs the telomere length and molecular change at RNA and protein level by RNA-seq and proteomics. Then we identified the potential interacting proteins with UBQLN1 using immunoprecipitation-mass spectrometry (IP-MS). Furthermore, the potential mechanisms underlying the shortened telomeres in UBQLN1-deficient hESCs were analyzed.ResultsWe show that Ubiquilin1 (UBQLN1) is critical for telomere maintenance in human embryonic stem cells (hESCs) via promoting mitochondrial function. UBQLN1 deficiency leads to oxidative stress, loss of proteostasis, mitochondria dysfunction, DNA damage, and telomere attrition. Reducing oxidative damage and promoting mitochondria function by culture under hypoxia condition or supplementation with N-acetylcysteine partly attenuate the telomere attrition induced by UBQLN1 deficiency. Moreover, UBQLN1 deficiency/telomere shortening downregulates genes for neuro-ectoderm lineage differentiation.ConclusionsAltogether, UBQLN1 functions to scavenge ubiquitinated proteins, preventing their overloading mitochondria and elevated mitophagy. UBQLN1 maintains mitochondria and telomeres by regulating proteostasis and plays critical role in neuro-ectoderm differentiation.

Hippocampal aggregation signatures of pathogenic UBQLN2 in amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic variants in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia characterized by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial amyotrophic lateral sclerosis and/or frontotemporal dementia cases not caused by UBQLN2 pathogenic variants, particularly C9orf72-linked cases. This makes the mechanistic role of mutant ubiquilin 2 protein and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 44 genotypically diverse amyotrophic lateral sclerosis cases with or without frontotemporal dementia, including eight cases with UBQLN2 variants [resulting in p.S222G, p.P497H, p.P506S, p.T487I (two cases) and p.P497L (three cases)]. Using multiplexed (five-label) fluorescent immunohistochemistry, we mapped the co-localization of ubiquilin 2 with phosphorylated TDP-43, dipeptide repeat aggregates and p62 in the hippocampus of controls (n = 6), or amyotrophic lateral sclerosis with or without frontotemporal dementia in sporadic (n = 20), unknown familial (n = 3), SOD1-linked (n = 1), FUS-linked (n = 1), C9orf72-linked (n = 5) and UBQLN2-linked (n = 8) cases. We differentiate between (i) ubiquilin 2 aggregation together with phosphorylated TDP-43 or dipeptide repeat proteins; and (ii) ubiquilin 2 self-aggregation promoted by UBQLN2 pathogenic variants that cause amyotrophic lateral sclerosis and/or frontotemporal dementia. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wild-type ubiquilin 2 in amyotrophic lateral sclerosis with or without frontotemporal dementia, whereby mutant ubiquilin 2 is more prone than wild-type to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

Humoral immune response to tumor-associated antigen Ubiquilin 1 (UBQLN1) and its tumor-promoting potential in lung cancer

BackgroundThis study aims to investigate the expression of UBQLN1 in lung cancer (LC) tissue and the diagnostic capability of autoantibody to UBQLN1 (anti-UBQLN1) in the detection of LC and the discrimination of pulmonary nodules (PNs).MethodsSera from 798 participants were used to discover and validate the level of autoantibodies via HuProt microarray and Enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis was applied to establish model. Receiver operating characteristic curve (ROC) analysis was performed to evaluate the diagnostic potential. Immunohistochemistry was performed to detect UBQLN1 expression in 88 LC tissues and 88 para-tumor tissues. qRT-PCR and western blotting were performed to detect the expression of UBQLN1 at the mRNA and protein levels, respectively. Trans-well assay and cell counting kit-8 (CCK-8) was used to investigate the function of UBQLN1.ResultsAnti-UBQLN1 was identified with the highest fold change by protein microarray. The level of anti-UBQLN1 in LC patients was obviously higher than that in NC or patients with benign lung disease of validation cohort 1 (P<0.05). The area under the curve (AUC) of anti-UBQLN1 was 0.610 (95%CI: 0.508-0.713) while reached at 0.822 (95%CI: 0.784-0.897) when combining anti-UBQLN1 with CEA, CYFRA21-1, CA125 and three CT indicators (vascular notch sign, lobulation sign and mediastinal lymph node enlargement) in the discrimination of PNs. UBQLN1 protein was overexpressed in lung adenocarcinoma (LUAD) tissues compared to para-tumor tissues. UBQLN1 knockdown remarkably inhibited the migration, invasion and proliferation of LUAD cell lines.ConclusionsAnti-UBQLN1 might be a potential biomarker for the diagnosis of LC and the discrimination of PNs.

Distribution of ubiquilin 2 and TDP-43 aggregates throughout the CNS in UBQLN2 p.T487I-linked amyotrophic lateral sclerosis and frontotemporal dementia.

Mutations in the UBQLN2 gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The neuropathology of such UBQLN2-linked cases of ALS/FTD is characterised by aggregates of the ubiquilin 2 protein in addition to aggregates of the transactive response DNA-binding protein of 43 kDa (TDP-43). ALS and FTD without UBQLN2 mutations are also characterised by TDP-43 aggregates, that may or may not colocalise with wildtype ubiquilin 2. Despite this, the relative contributions of TDP-43 and ubiquilin 2 to disease pathogenesis remain largely under-characterised, as does their relative deposition as aggregates across the central nervous system (CNS). Here we conducted multiplex immunohistochemistry of three UBQLN2 p.T487I-linked ALS/FTD cases, three non-UBQLN2-linked (sporadic) ALS cases, and 8 non-neurodegenerative disease controls, covering 40 CNS regions. We then quantified ubiquilin 2 aggregates, TDP-43 aggregates and aggregates containing both proteins in regions of interest to determine how UBQLN2-linked and non-UBQLN2-linked proteinopathy differ. We find that ubiquilin 2 aggregates that are negative for TDP-43 are predominantly small and punctate and are abundant in the hippocampal formation, spinal cord, all tested regions of neocortex, medulla and substantia nigra in UBQLN2-linked ALS/FTD but not sporadic ALS. Curiously, the striatum harboured small punctate ubiquilin 2 aggregates in all cases examined, while large diffuse striatal ubiquilin 2 aggregates were specific to UBQLN2-linked ALS/FTD. Overall, ubiquilin 2 is mainly deposited in clinically unaffected regions throughout the CNS such that symptomology in UBQLN2-linked cases maps best to the aggregation of TDP-43.

Platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury.

The effect of platelet-rich plasma on nerve regeneration remains controversial. In this study, we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with platelet-rich plasma. Twenty-eight rabbits were divided into the following groups (7 rabbits/group): model, low-concentration PRP (2.5-3.5-fold concentration of whole blood platelets), medium-concentration PRP (4.5-6.5-fold concentration of whole blood platelets), and high-concentration PRP (7.5-8.5-fold concentration of whole blood platelets). Electrophysiological and histomorphometrical assessments and proteomics analysis were used to evaluate regeneration of the sciatic nerve. Our results showed that platelet-rich plasma containing 4.5-6.5- and 7.5-8.5-fold concentrations of whole blood platelets promoted repair of sciatic nerve injury. Proteomics analysis was performed to investigate the possible mechanism by which platelet-rich plasma promoted nerve regeneration. Proteomics analysis showed that after sciatic nerve injury, platelet-rich plasma increased the expression of integrin subunit β-8 (ITGB8), which participates in angiogenesis, and differentially expressed proteins were mainly enriched in focal adhesion pathways. Additionally, two key proteins, ribosomal protein S27a (RSP27a) and ubiquilin 1 (UBQLN1), which were selected after protein-protein interaction analysis, are involved in the regulation of ubiquitin levels in vivo. These data suggest that platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury by affecting angiogenesis and intracellular ubiquitin levels.

Abnormally elevated ubiquilin‑1 expression in breast cancer regulates metastasis and stemness via AKT signaling.

Ubiquilin-1 (UBQLN1) is an essential factor for the maintenance of proteostasis in cells. It is important for the regulation of different protein degradation mechanisms, including the ubiquitin-proteasome system, autophagy and endoplasmic reticulum-associated protein degradation pathways. However, the role of UBQLN1 in cancer progression remains largely unknown. In the present study, the expression, functions and molecular mechanisms of UBQLN1 in breast cancer tissue samples and cell lines were explored. Immunohistochemical and bioinformatics analyses revealed that UBQLN1 expression was significantly upregulated in breast cancer tissues and cell lines. UBQLN1 expression in breast cancer was significantly associated with lymph node metastasis and TNM stage. Moreover, a high UBQLN1 expression was a predictor of an unfavorable survival in patients with breast cancer. In vitro, UBQLN1 silencing markedly inhibited cell migration and invasion, epithelial-to-mesenchymal transition (EMT) and MMP expression. UBQLN1 silencing attenuated the stem cell-like properties of breast cancer cells, including their mammosphere-forming abilities. UBQLN1 knockdown also enhanced breast cancer cell chemosensitivity to paclitaxel. The expression levels of the stem cell markers. Aldehyde dehydrogenase 1 (ALDH1), Oct-4 and Sox2 were significantly decreased in the cells in which UBQLN1 was silenced, whereas breast cancer stem cells exhibited an increased expression of UBQLN1. Mechanistically, UBQLN1 knockdown inhibited the activation of AKT signaling, as revealed by the increased PTEN expression and the decreased expression of phosphorylated AKT in cells in which UBQLN1 was silenced. On the whole, the present study demonstrates that UBQLN1 is aberrantly upregulated in breast cancer and predicts a poor prognosis. The silencing of UBQLN1 inhibited the invasion, EMT and stemness of breast cancer cells, possibly via AKT signaling.

Ubiquilin 1 suppresses the cancer stem cell–like traits of non-small cell lung cancer cells by regulating reactive oxygen species homeostasis

ABSTRACT Cancer stem cell (CSC) has been confirmed to trigger tumor occurrence and progression and CSC can develop strategies to maintain a lower reactive oxygen species (ROS) level compared to cancer cells. However, the mechanisms contributing to ROS homeostasis in CSC are still lacking key elements. In the current study, we found that reductive redox states and ROS levels were suppressed in non-adherent spheres formed by non-small cell lung cancer (NSCLC) cells, which were confirmed to hold CSC-like traits. However, mitochondria DNA content and cellular oxygen consumption rate analyses revealed fewer numbers of mitochondria in NSCLC spheres. Further exploration attributed this result to decreased mitochondrial biogenesis, likely resulted from the accelerated degradation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). Mechanistic studies indicated that Ubiquilin 1 (UBQLN1) increased PGC1α protein stability via reducing the ubiquitination of PGC1α protein. Moreover, UBQLN1 was lowly expressed in NSCLC spheres compared to that in parental NSCLC cells and UBQLN1 overexpression suppressed the CSC-like traits of NSCLC cells, which was characterized as the decrease of ALDH1 activity, sphere-formation ability, and CSC marker expression. Finally, clinical investigations further demonstrated that UBQLN1 level was positively correlated with patient’s survival of lung adenocarcinoma, but not squamous cell carcinoma of lung. Taken together, our results revealed a novel mechanism involving ROS homeostasis and mitochondrial biogenesis in non-small cell lung CSCs, which may provide novel potential targets and methods for NSCLC patients.

MiR-337-5p promotes the development of cardiac hypertrophy by targeting Ubiquilin-1 (UBQLN1)

ABSTRACT Cardiac hypertrophy is an adaptive response of the myocardium to the pressure overload of the heart. MicroRNAs (miRNAs/miRs) are shown to be directly involved in the development of cardiac hypertrophy. However, the function of miR-337-5p and its potential contribution to the serine/threonine-protein kinase, a mammalian target of rapamycin (mTOR) signaling in cardiac hypertrophy remains unknown. In the present study, miR-337-5p expression was examined in cardiomyocytes treated with angiotensin II (Ang II). An adenovirus vector system was employed to knockdown miR-337-5p expression to investigate its functions in cardiac hypertrophy. The results revealed a significant increase in the expression of miR-337-5p in cardiomyocytes treated with Ang II as compared with controls. In addition, downregulation of miR-337-5p expression inhibited cardiac hypertrophy both in vitro and in vivo. Dual-luciferase reporter assays demonstrated Ubiquilin-1 (UBQLN1) as the direct target of miR-337-5p, and revealed its function in the modulation of mTOR signaling. Rescue experiments indicated that UBQLN1 overexpression reversed the effects of miR-337-5p, and further verified this interaction. In summary, the results of the present study show that miR-337-5p silencing attenuates cardiac hypertrophy by targeting UBQLN1. Therefore, miR-337-5p plays a critical role in cardiac hypertrophy and may serve as a new therapeutic target.

The impacts of ubiquilin 1 (UBQLN1) knockdown on cells viability, proliferation, and apoptosis are mediated by p53 in A549 lung cancer cells.

BackgroundLittle is known about the relationship between ubiquilin 1 (UBQLN1) and p53, both of them have been implicated in the development and progression of non-small cell lung cancer (NSCLC). In this study, we aimed to explore the role of loss of UBQLN1 in cell viability and proliferation, and cell apoptosis in human lung adenocarcinoma A549 cells.MethodsCell viability, proliferation, and apoptosis were determined by MTT, BrdU, and TUNEL assays, respectively. Adenoviruses carrying cDNA or siRNA were used to overexpress or silence target protein. Dihydroethidium (DHE) staining was performed to measure the real-time formation of intracellular reactive oxygen species (ROS). The chymotrypsin-like activity of 20S proteasome core was determined by using synthetic fluorogenic peptide substrate.ResultsUBQLN1 silencing led to a reduction of p53 protein levels and overexpression of p53 reversed the effects of UBQLN1 knockdown (KD) on cell viability, proliferation, and apoptosis. Furthermore, deficiency of UBQLN1 activated autophagy activity but did not affect proteasome activity. Inhibition of autophagy restored p53 protein levels in UBQLN1-KD A549 cells. In addition, UBQLN1 KD markedly inhibited phosphorylation of mammalian target of rapamycin (mTOR) and its downstream ribosomal S6 kinase (S6K).ConclusionsOur experiments suggested that the regulation of UBQLN1 on cell viability, proliferation, and apoptosis was mediated by mTOR/autophagy/p53 signaling pathway.

Early-onset of Frontotemporal Dementia and Amyotrophic Lateral Sclerosis in an Albanian Patient with a c.1319C&gt;T Variant in the UBQLN2 Gene

Background: Frontotemporal Dementia (FTD) is the second most common cause of dementia under 65 years of age; it has a prevalence of 4-15 per 100,000 persons. The overt disease usually manifests in the sixth decade, and it is extremely rare to find affected patients in their twenties. Objective: Here, we present the clinical and molecular genetic findings of an Albanian family with a patient with early-onset FTD and Amyotrophic Lateral Sclerosis (ALS). Methods: Given the great variability of clinical presentation of FTD and the number of genes involved, targeted Next Generation Sequencing (NGS) was used to screen the DNA of the 27-year-old male patient. Segregation analysis was performed in available family members. Results and Discussion: A variant, consisting of a proline-leucine amino acid substitution in position 440, was identified in the UBQLN2 gene on the X-chromosome. This variant was previously reported as a variant of unknown significance in a 30-year-old female patient with amyotrophic lateral sclerosis. With the description of our case, we add evidence on its involvement, also in ALS-FTD. The variant is in a functional domain important for interaction with HSP70 and this, in turn, may impair the shuttling of proteins to the proteasome leading to an accumulation of protein aggregates. The variant was inherited from the unaffected mother, in line with the fact that incomplete penetrance has been widely described for this gene. Conclusion:The present report adds information regarding one of 34 variants in the UBQLN2 gene reported so far in association with neurodegeneration and proposes a molecular pathogenesis of ALS-FTD in this patient.

Amyotrophic lateral sclerosis-linked UBQLN2 mutants inhibit endoplasmic reticulum to Golgi transport, leading to Golgi fragmentation and ER stress.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that are related genetically and pathologically. Mutations in the UBQLN2 gene, encoding the ubiquitin-like protein ubiquilin2, are associated with familial ALS/FTD, but the pathophysiological mechanisms remain unclear. Here, we demonstrate that ALS/FTD UBQLN2 mutants P497H and P506T inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in neuronal cells. In addition, we observed that Sec31-positive ER exit sites are clustered in UBQLN2T487I patient spinal cord tissues. Both the ER-Golgi intermediate (ERGIC) compartment and the Golgi become disorganised and fragmented. This activates ER stress and inhibits ER-associated degradation. Hence, this study highlights perturbations in secretory protein trafficking and ER homeostasis as pathogenic mechanisms associated with ALS/FTD-associated forms of UBQLN2.

Substitution 3A protein of foot-and-mouth disease virus of attenuated ZB strain rescued the viral replication and infection in bovine cells

The non-structural protein 3A of foot-and-mouth disease virus (FMDV) plays an important role in viral replication, virulence and determination of host range. Previously we identified genomic changes in gene encoding 3A protein between the attenuated ZBatt strain and its parental virulent strain during the attenuation process. However, the effects of changes in 3A protein on viral replication and infection of the rabbit-attenuated ZBatt virus during the attenuation process are poorly understood. In this study, a chimeric virus, rZBatt-3A, was constructed by introducing the 3A gene of virulent ZB virus into its attenuated vaccine ZB strain. Subsequently, the biological characteristics between rZBatt-3A and its parental virus (rZBatt) were compared. The relative expression level of four host cell proteins that interact with FMDV 3A were also analyzed. The results showed that the chimeric virus rZBatt-3A exhibited significantly different growth properties and plaque phenotypes from rZBatt in primary fetal bovine kidney (BK) cells. Cytopathic effect (CPE) of the rZBatt-3A was observed in BK cells with smaller plaque size, but CPE from the rZBatt could not be observed. The viral RNA replication was higher in rZBatt-3A-infected BK cells than in rZBatt-infected cells at 24 hpi (P < .05). In addition, the relative mRNA expression level of Ubiquilin 1 (UBQLN1) was significantly increased in rZBatt-3A-infected BK cells than in rZBatt-infected cells (P < .01) suggesting that UBQLN1 may be associated with 3A protein changes. Thus, the substitution of 3A protein altered the replication efficiency of attenuated ZB virus in bovine cells. Our data suggested that changes in 3A protein might be associated with the attenuation of ZB virus, which shed more lights in molecular mechanisms about attenuation of FMDV.

Genetic alterations of C9orf72, SOD1, TARDBP, FUS, and UBQLN2 genes in patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis is the most common motor neuron disease of the adulthood. Genetic analyses performed on cases with sporadic ALS (sALS) and familial ALS (fALS) have revealed mutations most commonly in the genes C9orf72, SOD1, TARDBP, FUS, and UBQLN2. The aim of this study was to investigate the presence and incidence of these most common genomic alterations in these genes of 30 ALS cases. Increase in the number of hexanucleotide repeats within the gene C9orf72 was investigated using the fragment analysis method. A heterozygote c.-45 + 162_-45 + 163insGGGGCC alteration was observed in the first intron of the C9orf72 gene in only two sALS cases (6.6%), c.72 + 133C>T alteration was observed in the first intron of the SOD1 gene in six fALS and two sALS cases (26.6%), a c.169 + 41C>A alteration was observed in the second intron of the same gene in one case, a c.239 + 34A>C alteration was observed in the third intron of the same gene in three fALS cases (10%), and a c.714 + 67_714 + 68insG alteration was observed in the fifth intron of the TARDBP gene in six sALS cases (20%). Three different genomic alterations were detected in the FUS gene; two silent variants p.G49G (c.147C>A) in 3 cases (10%) and p.Y97Y (c.291C>T) in 25 cases (83%) and an intronic variant c.1067-61T˃C in 4 cases (13%). A novel missense variant, p.G457H (c.1371G>C), was observed in the UBQLN2 gene in one case with sALS. Our study has revealed three novel and previously reported genomic alterations in the ALS-related genes.

Clinical significance of ubiquilin 1 in gastric cancer.

Ubiquilin 1 (UBQLN1) plays an essential role in the regulation of protein degradations which is involved in the pathophysiology of neurodegenerative diseases and cancer. This study aimed to investigate the expression level of UBQLN1 in gastric cancer and evaluated the relationship between its expression and clinicopathological characteristics, as well as prognostic of patients with gastric cancer. Immunohistochemistry (IHC) was used to detect the expression levels of UBQLN1 in 179 pairs of gastric cancer and adjacent normal tissues. The UBQLN1 was significantly upregulated in gastric cancer tissue. High UBQLN1 expression was associated with high histological grade, invasion, lymph node metastasis, and tumor node metastasis (TNM) stage III (P < .001). Multivariate Cox analysis showed that larger tumor size (HR = 3.125, 95%CI: 2.031–4.808, P < .001), histological grade 3 (HR = 15.313, 95%,CI: 8.075–29.041, P < .001), pT3 + pT4 (HR = 3.224, 95%CI: 1.389–7.483, P = .006), LNM (HR = 4.467, 95%CI: 2.404–8.302, P < .001), TNM stage III (HR = 2.152, 95%CI: 1.289–3.594, P = .003), and high UBQLN1 expression (HR = 2.547, 95%CI: 1.511–4.292, P < .001) were significantly associated with worse prognosis of patients with gastric cancer. In conclusion, high UBQLN1 expression was an independent worse prognostic factor for patients with gastric cancer.

A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. We herein performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. We found that the depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. Our results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to our understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.

Regulation of insulin-like growth factor receptors by Ubiquilin1.

Insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase that mediates growth, proliferation and survival. Dysregulation of IGF pathway contributes to the initiation, progression and metastasis of cancer and is also involved in diseases of glucose metabolism, such as diabetes. We have identified Ubiquilin1 (UBQLN1) as a novel interaction partner of IGF1R, IGF2R and insulin receptor (INSR). UBQLN family of proteins have been studied primarily in the context of protein quality control and in the field of neurodegenerative disorders. Our laboratory discovered a link between UBQLN1 function and tumorigenesis, such that UBQLN1 is lost and underexpressed in 50% of human lung adenocarcinoma cases. We demonstrate here that UBQLN1 regulates the expression and activity of IGF1R. Following loss of UBQLN1 in lung adenocarcinoma cells, there is accelerated loss of IGF1R. Despite decreased levels of total receptors, the ratio of active : total receptors is higher in cells that lack UBQLN1. UBQLN1 also regulates INSR and IGF2R post-stimulation with ligand. We conclude that UBQLN1 is essential for normal regulation of IGF receptors. UBQLN-1-deficient cells demonstrate increased cell viability compared with control when serum-starved and stimulation of IGF pathway in these cells increased their migratory potential by 3-fold. As the IGF pathway is involved in processes of normal growth, development, metabolism and cancer progression, understanding its regulation by Ubiquilin1 can be of tremendous value to many disciplines.

The STI and UBA Domains of UBQLN1 Are Critical Determinants of Substrate Interaction and Proteostasis.

There are five Ubiquilin proteins (UBQLN1-4, UBQLN-L), which are evolutionarily conserved and structurally similar. UBQLN proteins have three functional domains: N-terminal ubiquitin-like domain (UBL), C-terminal ubiquitin-associated domain (UBA), and STI chaperone-like regions in the middle. Alterations in UBQLN1 gene have been detected in a variety of disorders ranging from Alzheimer's disease to cancer. UBQLN1 has been largely studied in neurodegenerative disorders in the context of protein quality control. Several studies have hypothesized that the UBA domain of UBQLN1 binds to poly-ubiquitin chains of substrate and shuttles it to the proteasome via its UBL domain for degradation. UBQLN1 either facilitates degradation (Ataxin3, EPS15) or stabilizes (PSEN1/2, BCLb) substrates it binds to. The signal that determines this fate is unknown and there is conflicting data to support the existing working model of UBQLN1. Using BCLb as a model substrate, we characterized UBQLN1-substrate interaction. We identified the first two STI domains of UBQLN1 as critical for binding to BCLb. Interaction of UBQLN1 with BCLb is independent of ubiquitination of BCLb, but interaction with ubiquitin via UBA domain is required for stabilization of BCLb. Similarly, we showed that UBQLN1 interacts with IGF1R and ESYT2 through the STI domains and stabilizes these proteins through its UBA domain. Interactions that are not dependent on STI domains, for example, UBL mediated interaction with PSMD4 and BAG6, do not appear to be stabilized by UBQLN1. We conclude that fate of substrates that UBQLN1 associates with, is interaction domain specific. J. Cell. Biochem. 118: 2261-2270, 2017. © 2017 Wiley Periodicals, Inc.

The mir-675-5p regulates the progression and development of pancreatic cancer via the UBQLN1-ZEB1-mir200 axis.

Pancreatic cancer (PC) is a highly lethal disease due to extensive metastatic lesions. Accumulating evidence suggests that miR-675-5p plays different roles in metastasis through the regulation of epithelial to mesenchymal (EMT) and the mesenchymal to epithelial transitions (MET) in different cancers. ZEB1 promotes the EMT process by controlling the expression of E-cadherin and may have a reciprocal regulation with Ubiquilin1 (UBQLN1) and mir-200 family in cancer progression. In the present study, we showed that decreased expression of miR-675-5p is associated with the enhanced cell proliferation and survival of PC cells, while the increased expression of mir-675-5p shows the opposite one. The mir-675-5p could decrease the expression of mir-200 which is intermediated by ZEB1, and increase the expression of UBQLN1 gene. The mir-675-5p can increase the expression of ZEB1 mRNA, but the ZEB1 protein level was decreased. When mir-675-5p mimics and siUBQLN1 were co-transfected into the pancreatic cancer Patu8988 cells, the expression of ZEB1 protein was increased. It suggests that mir-675-5p may affect ZEB1 in a post-transcriptional level which was verified to be regulated by UBQLN1 protein. Hence, mir-675-5p regulates the progression of pancreatic cancer cells through the UBQLN1-ZEB1-mir200 pathway.

No Evidence for Pathogenic Role of UBQLN2 Mutations in Sporadic Amyotrophic Lateral Sclerosis in the Mainland Chinese Population

Mutations in the UBQLN2 gene, which encodes a member of the ubiquitin-like protein family (ubiquilin-2), have been identified in patients with dominant X-linked amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal dementia (FTD). We analyzed mutations in the UBQLN2 gene in a Chinese cohort of 515 patients with sporadic ALS (sALS). A novel missense mutation (p.M392V) was detected in one sALS patient. The p.M392V mutation substitutes a highly conserved residue, has not been reported in the population databases, and previously, at the same residue, a missense mutation p.M392I was detected in two Turkey ALS patients and was considered to be pathogenic, so the M392V is a variant of uncertain significance (VOUS) for ALS. We also found a deletion mutation (p.P500_G502del), which seems to be benign. In conclusion, our data suggest that mutations in the UBQLN2 gene are rare in Chinese sALS patients.

PUB131 MIR155 Mediated Down-Regulation of Ubiquilin 1 Induces EMT in Lung Cancer Cells

Ubiquilin 1 (UBQLN1) functions as an adaptor protein that links the ubiquitination machinery to the proteasome for protein degradation. However, recently our lab has shown that despite the role in protein degradation, UBQLN1 also plays a role in cancer biology. In the present study, we have examined the post-transcriptional regulation of UBQLN1 and identified a miRNA responsible for regulation of UBQLN1 in A549 and H358 cells. In silico approaches involve the use of TargetScan and miRDB web portals for identification of miRNA sites in 3'-UTR of UBQLNs. Transfections of miRNA mimics and inhibitors were performed using Dhafrmafect reagent. Epithelial to mesenchymal transformation (EMT) was assayed by colony formation, scratch assay and cell migration and cell invasion assays. Dual luciferase assay was carried out to validate the binding of MIR155 on 3'-UTR of UBQLN1. In silico approaches suggested miR-200, miR-425 and miR-155 as probable miRNAs capable of regulating levels of UBQLN1. However, over-expression of only miR-155 significantly down-regulated levels of UBQLN1 protein in A549 and H358, two different lung adenocarcinoma cell lines. Interestingly, introduction of miR-155 significantly increased the proliferation of A549 and H358 cells. Flow cytometry based cell cycle analysis of miR-155 over-expressing cells showed a significant decrease in the G1 phase of the cell cycle. Over-expression of miR-155 induced colony formation, cell invasion and migration of A549 and H358 cells. Further, dual luciferase assays carried out with 3’-UTR of UBQLN1 and miR-155 mimic confirmed in vivo binding capability of miR-155 on 3’-UTR of UBQLN1. Moreover, when MIR155 transfected A549 cells are injected in immunodeficient mice they colonize and grow faster in lungs of mice than non-targeting control transfected A549 cells. In conclusion, for the first time our studies have identified a miRNA that regulates UBQLN1 in lung carcinoma cells.