Ubiquitin-conjugating Enzyme E2 L3 Research Articles

Role of Ubiquitin-conjugating enzyme E2 (UBE2) in two immune-mediated inflammatory skin diseases: a mendelian randomization analysis

Psoriasis vulgaris (PV) and Atopic dermatitis (AD) are the two major types of immune-mediated inflammatory skin disease (IMISD). Limited studies reported the association between Ubiquitin-conjugating enzyme E2 (UBE2) and IMISD. We employed a two-sample Mendelian randomization (MR) study to assess the causality between UBE2 and PV & AD. UBE2 association genome-wide association study (GWAS) data were collected. The inverse variance weighted (IVW) method was utilized as the principal method in our Mendelian randomization (MR) study, with additional using the MR-Egger, weighted median, simple mode, and weighted mode methods. The MR-Egger intercept test, Cochran’s Q test, MR-Pleiotropy RESidual Sum and Outlier (MR-PRESSO) and leave-one-out analysis were conducted to identify heterogeneity and pleiotropy, colocalization analysis was also performed. The results showed that Ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) was causally associated with PV (OR = 0.909, 95% CI: 0.830–0.996, P = 0.040), Ubiquitin-conjugating enzyme E2 L3 (UBE2L3) was causally associated with AD (OR = 0.799, 95% CI: 0.709–0.990, P < 0.001). Both UBE2V1 and UBE2L3 may play protective roles in patients with PV or AD, respectively. No other significant result has been investigated. No heterogeneity or pleiotropy was observed. This study provided new evidence of the relationship between UBE2V1 and PV, UBE2L3 and AD. Our MR suggested that UBE2V1 plays an inhibitory role in PV progression, UBE2L3 plays an inhibitory role in AD. These could be novel and effective ways to treat PV and AD.

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

UBE2L3 promotes squamous cell carcinoma progression in the oral cavity and hypopharynx via activating the NF-κB signaling by increasing IκBα degradation.

Oral squamous cell carcinoma (OSCC) and hypopharyngeal squamous cell carcinoma (HSCC) are representative of head and neck squamous cell carcinoma (HNSCC) and the molecular pathogenesis has not been completely clarified. Ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is the key member of the E2 family that encodes 153 amino acid residues. Previous studies demonstrate that UBE2L3 is aberrantly overexpressed in various types of human cancers, suggesting that UBE2L3 may function as an oncogene. However, its functional role and the potential mechanisms in the OSCC and HSCC remain unclear. In the present study, we found that UBE2L3 was significantly upregulated in clinical HNSCC samples and HNSCC cell lines, and patients with lower UBE2L3 expression have a higher survival rate. Two HNSCC cell lines FaDu (HSCC cells) and CAL-27 (OSCC cells) with moderate expression of UBE2L3 were selected for in vitro experiments. We proved that UBE2L3 overexpression was positively associated with cellular malignant phenotypes in vitro, including proliferation, invasion, migration, and tumor growth in vivo. Conversely, UBE2L3 suppression diametrically yielded opposing results. Our further study demonstrated that overexpression of UBE2L3 significantly activated the nuclear factor kappa B (NF-κB) signaling pathway through promoting NF-κB p65 nuclear translocation and the ubiquitination and degradation of IκBα protein. Additionally, UBE2L3 was proved to be targeted and negatively regulated by miR-378a-5p, and UBE2L3 overexpression reversed the effects of miR-378a-5p upregulation. Collectively, the present study indicates that UBE2L3 may promote OSCC and HSCC progression via activating the NF-κB signaling by increasing IκBα degradation, indicating that UBE2L3 may be a potential therapeutic target for the treatment of HNSCC.

Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization.

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

Overexpression of ubiquitin-conjugating enzyme E2 L3 in hepatocellular carcinoma potentiates apoptosis evasion by inhibiting the GSK3β/p65 pathway.

UBE2L3 is a ubiquitin-conjugating protein belonging to the E2 family that consists of 153 amino acid residues. In this study, we found that UBE2L3 was generally upregulated in clinical HCC samples compared to non-tumour samples and that there was a strong association between high UBE2L3 expression and tumour size, clinical grade and prognosis in HCC patients. UBE2L3 depletion inhibited the proliferation and induced the apoptosis of HCC cells. At the molecular level, we observed that UBE2L3 depletion enhanced the protein stability of GSK3β, thus promoting the expression and activation of GSK3β. Subsequently, activated GSK3β phosphorylated p65 and promoted its nuclear translocation to increase the expression of target genes, including PUMA, Bax, Bim, Bad, and Bid. In vivo, knockout of UBE2L3 in HCC cells inhibited tumour growth in orthotopic liver injection nude mouse models. Moreover, inhibition of p65 or GSK3β significantly restored the effects induced by UBE2L3 knockout in HCC. Together, this study reveals the stimulatory effect of UBE2L3 on HCC cell proliferation, suggesting that UBE2L3 may be an important pro-tumorigenic factor in liver carcinogenesis and a potential therapeutic target of HCC.

USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy

ABSTRACTPRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged mitochondria. Deubiquitinating enzymes (DUBs) functionally reversing PRKN ubiquitination are critical in controlling the magnitude of PRKN-mediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its pro-mitophagy effect remains to be identified and characterized. Here, we demonstrated that USP33/VDU1 is localized at the outer membrane of mitochondria and serves as a PRKN DUB through their interaction. Cellular and in vitro assays illustrated that USP33 deubiquitinates PRKN in a DUB activity-dependent manner. USP33 prefers to remove K6, K11, K48 and K63-linked ubiquitin conjugates from PRKN, and deubiquitinates PRKN mainly at Lys435. Mutation of this site leads to a significantly decreased level of K63-, but not K48-linked PRKN ubiquitination. USP33 deficiency enhanced both K48- and K63-linked PRKN ubiquitination, but only K63-linked PRKN ubiquitination was significantly increased under mitochondrial depolarization. Further, USP33 knockdown increased both PRKN protein stabilization and its translocation to depolarized mitochondria leading to the enhancement of mitophagy. Moreover, USP33 silencing protects SH-SY5Y human neuroblastoma cells from the neurotoxin MPTP-induced apoptotic cell death. Our findings convincingly demonstrate that USP33 is a novel PRKN deubiquitinase antagonizing its regulatory roles in mitophagy and SH-SY5Y neuron-like cell survival. Thus, USP33 inhibition may represents an attractive new therapeutic strategy for PD patients.Abbreviations: CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DUB: deubiquitinating enzymes; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OMM: outer mitochondrial membrane; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; TM: transmembrane; Ub: ubiquitin; UBA1: ubiquitin like modifier activating enzyme 1; UBE2L3/UbcH7: ubiquitin conjugating enzyme E2 L3; USP33: ubiquitin specific peptidase 33; WT: wild type.

A Functional Variant in Ubiquitin Conjugating Enzyme E2 L3 Contributes to Hepatitis B Virus Infection and Maintains Covalently Closed Circular DNA Stability by Inducing Degradation of Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3A.

Hepatitis B virus (HBV) infection is a common infectious disease, in which nuclear covalently closed circular DNA (cccDNA) plays a key role in viral persistence, viral reactivation after treatment withdrawal, and drug resistance. A recent genome-wide association study has identified that the ubiquitin conjugating enzyme E2 L3 (UBE2L3) gene is associated with increased susceptibility to chronic HBV (CHB) infection in adults. However, the association between UBE2L3 and children with CHB and the underlying mechanism remain unclear. In this study, we performed two-stage case-control studies including adults and independent children in the Chinese Han population. The rs59391722 allele in the promoter of the UBE2L3 gene was significantly associated with HBV infection in both adults and children, and it increased the promoter activity of UBE2L3. Serum UBE2L3 protein levels were positively correlated with HBV viral load and hepatitis B e antigen (HBeAg) levels in children with CHB. In an HBV infection cell model, UBE2L3 knockdown significantly reduced total HBV RNAs, 3.5-kb RNA, as well as cccDNA in HBV-infected HepG2-Na+ /taurocholate cotransporting polypeptide cells and human primary hepatocytes. A mechanistic study found that UBE2L3 maintained cccDNA stability by inducing proteasome-dependent degradation of apolipoprotein B mRNA editing enzyme catalytic subunit 3A, which is responsible for the degradation of HBV cccDNA. Moreover, interferon-α (IFN-α) treatment markedly decreased UBE2L3 expression, while UBE2L3 silencing reinforced the antiviral activity of IFN-α on HBV RNAs, cccDNA, and DNA. rs59391722 in UBE2L3 was correlated with HBV DNA suppression and HBeAg loss in response to IFN-α treatment of children with CHB. Conclusion: These findings highlight a host gene, UBE2L3, contributing to the susceptibility to persistent HBV infection; UBE2L3 may be involved in IFN-mediated viral suppression and serve as a potential target in the prevention and treatment of HBV infection.

The butterfly effect in viral infection: From a host DNA single nucleotide change to HBV episome steadiness

Chronic hepatitis B virus (HBV) infection is one of the major health care burdens throughout the world. The chronicity of HBV infection is supported by the intracellular persistence of a multicopy viral episome called covalently closed circular DNA (cccDNA), which is the bona fide viral transcription template and resistant to currently available antivirals. 1 Therefore, noncytolytic elimination of cccDNA is considered as the “holy grail” for a cure of hepatitis B, and the metabolism and transcription regulation of cccDNA have become a hot topic in basic, translational, and clinical HBV research. 2 In a recent publication, Zhou et al conducted a case control study and identified a single nucleotide variant (SNV), rs59391722, located at −1128 bp of the promoter region of ubiquitin-conjugating enzyme E2L3 gene (UBE2L3, also called UBCH7), which is associated with susceptibility to HBV infection in children. 3 The SNVs at this site were found to be GG as a major SNV, and CC as a minor one, with transitional CG in Chinese Han population. GG and CG SNVs are associated with a higher binding efficiency of transcription activator NF-E2, leading to UBE2L3 upregulation compared to that in CC. Furthermore, via overexpression and knockdown approaches, the authors demonstrated that UBE2L3 facilitates HBV replication and antigen production primarily through maintaining cccDNA stability. Mechanistic studies revealed that UBE2L3 catalytically promotes the ubiquitin-dependent proteasomal degradation of a cellular cytidine deaminase APOBEC3A (A3A), which has been shown to cause base-pair mismatch hypermutations in cccDNA for apurinic/apyrimidinic endonuclease (APE1)-dependent degradation. 4 In addition, considering that A3A is an interferon-stimulated gene (ISG), the authors showed that UBE2L3 silencing could potentiate the antiviral activity of alpha-interferon (IFN-α) in cell culture model. Taken together, this study has further validated the function of A3A in editing and removing cccDNA, and established a proviral role of rs59391722-UBE2L3-A3A nexus in cccDNA persistence (Fig. 1). Figure 1 The rs59391722-UBE2L3-APOBEC3A nexus regulates HBV cccDNA stability. HBV establishes and maintains persistent infection in hepatocyte via the viral episomal cccDNA, which serves as transcription template to produce progeny virus and viral antigens (HBeAg, HBsAg, HBx). IFN-α treatment induces cccDNA degradation through ISG APOBEC3A (A3A)-mediated cccDNA cytidine deamination and subsequent APE1 cleavage. Zhou et al 3 identified a SNV rs59391722(C→G) that upregulates the expression of UBE2L3, which ubiquitinates A3A for proteasomal degradation, thereby maintaining the persistence of cccDNA and HBV infection. Figure 1 One of the strong sides of the work is that the authors analyzed HBV-associated SNVs from a small cohort of pediatric patients, as most chronic hepatitis B (CHB) patients acquire the infection through vertical transmission or during early childhood. 5 Besides the immunological dysfunction, the SNVs, such as rs59391722(C→G), may also contribute to the development of chronic HBV infection. Previous genome-wide association studies (GWASs) have largely focused on adult cohort and identified HBV-associated SNVs at 8 loci, including 7 HLA-related genes and UBE2L3, but lacked functional validations.6, 7, 8 The study by Zhou et al, for the first time, established the positive correlation of UBE2L3 expression with the clinical outcome of HBV infection in both adult and children patients; and delineated the UBE2L3-A3A axis as a novel regulatory mechanism of cccDNA longevity in cell culture infection models. This study has opened a new venue for future HBV research. It would be interesting to perform an extended study with wider sampling of CHB patients with different age and ethnic background to further justify the link between rs59391722 SNVs and HBV persistence. The co-evolution of HBV subtypes and SNVs patterns could also be assessed along with estimate of evolutionary advantage of the rs59391722 haplotypes. The UBE2L3-A3A axis can be further explored to identify additional viral/host factors that regulate cccDNA metabolism and function. One question would be whether HBV enhances the expression of UBE2L3 for its benefits. The answer might be yes because the authors showed that IFN-α downregulated UBE2L3 when HBV was inhibited by the cytokine. UBE2L3 is an E2 ubiquitin-conjugating enzyme, and even though it has been reported to have an E3-independent reactivity with lysine, 9 whether a specific E3 ubiquitin ligase, such as the UBE2L3-interacting UBE3A/E6AP, 10 is required for UBE2L3-mediated ubiquitination of A3A awaits further investigation. From the translational medicine perspective, the rs59391722(C→G) SNV may represent a risk factor for neonatal and childhood HBV infection, and the serum UBE2L3 may serve as a novel biomarker for monitoring the progression of hepatitis B and for prediction of response to IFN-α therapy. However, these potential diagnostic and prognostic values of rs59391722 and UBE2L3 should be further evaluated in larger cohort of patients. Since both cccDNA persistence and eradication largely rely on host functions, this study suggests that UBE2L3, or ideally the more-specific E3 ubiquitin ligase, if any, may be exploited as an antiviral target to promote A3A-mediated cccDNA decay and more importantly, to synergize IFN-α therapy.

Proteomics‐Based Investigations of Neglected and Tropical Diseases

Proteomics‐Based Investigations of Neglected and Tropical Diseases

Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis. A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels. These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.

Ubiquitin conjugating enzyme E2 L3 promoted tumor growth of NSCLC through accelerating p27kip1 ubiquitination and degradation.

The molecular pathogenesis of human lung cancer has not been completely clarified. Here, we reported that UBE2L3, a member of the ubiquitin-conjugating enzymes (E2s), were overexpressed in non-small-cell lung cancer (NSCLC) tissues compared with the non-tumor tissues. High expression of UBE2L3 was correlated with advanced tumor stage and adverse outcomes. Knockdown of UBE2L3 inhibited NSCLC cell growth while ectopic expression of UBE2L3 promoted NSCLC cell growth in a cell cycle dependent manner. The results of subcutaneous tumor xenograft studies revealed that knockdown of UBE2L3 attenuated the in vivo tumor growth. Mechanistically, we observed that UBE2L3 could interact with F-box protein Skp2, a member of the SCF (Skp2) ubiquitin ligase complex, and thus promoted the ubiquitination and proteasomal degradation of p27kip1. Furthermore, NSCLC cases with high level of UBE2L3 and low level of p27kip1 had worst prognosis, suggesting that combination of UBE2L3 and p27kip1 is a more powerful prognostic marker for NSCLC patients. Taken together, the current study presented a novel marker for predicting prognosis and a potential therapeutic target for NSCLC patients.

The haplotype of UBE2L3 gene is associated with Hashimoto's thyroiditis in a Chinese Han population.

BackgroundThe ubiquitin conjugating enzyme E2L3 (UBE2L3) gene is associated with susceptibility to many autoimmune diseases. The aim of this study was to investigate the association between UBE2L3 gene and autoimmune thyroid diseases (AITDs) and their clinical phenotypes.MethodsWe genotyped five single-nucleotide polymorphisms (SNPs) rs131654, rs5754217, rs2298428, rs140489 and rs5998672 of UBE2L3 gene in case groups including 1028 patients with AITDs [676 cases of Graves’ disease (GD) and 352 cases of Hashimoto’s thyroiditis (HT)] and control group including 897 healthy individuals.The genotyping was performed with the method of polymerase chain reaction-ligase detection reaction (PCR-LDR).ResultsThe frequencies of allele and genotype of five SNPs in gene UBE2L3 showed no statistically significant difference between case groups and control group, respectively. Moreover, no significant differences in frequencies of allele and genotype of five SNPs of the gene were found between clinical subphenotypes of AITDs and control group. Such subphenotypes included GD, HT, and thyroid associated ophthalmopathy (TAO). The negative results were also found in the frequency of other haplotypes of the gene except the haplotype of TCGGC, which was significantly higher in HT group than in control group (P = 0.031, OR = 1.441).ConclusionsThe present findings indicate that TCGGC haplotype is associated with an increased risk of HT and UBE2L3 gene is likely to be a susceptibility factor to HT in a Chinese Han population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12902-016-0098-6) contains supplementary material, which is available to authorized users.

Methylmercury can induce Parkinson's-like neurotoxicity similar to 1-methyl-4- phenylpyridinium: a genomic and proteomic analysis on MN9D dopaminergic neuron cells.

Exposure to environmental chemicals has been implicated as a possible risk factor for the development of neurodegenerative diseases. Our previous study showed that methylmercury (MeHg) exposure can disrupt synthesis, uptake and metabolism of dopamine similar to 1-methyl-4-phenylpyridinium (MPP(+)). The objective of this study was to investigate the effects of MeHg exposure on gene and protein profiles in a dopaminergic MN9D cell line. MN9D cells were treated with MeHg (1-5 μM) and MPP(+) (10-40 μM) for 48 hr. Real-time PCR Parkinson's disease (PD) arrays and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) were performed for the analysis. PD PCR array results showed that 19% genes were significantly changed in the 2.5 μM MeHg treated cells, and 39% genes were changed in the 5 μM MeHg treated cells. In comparison, MPP(+) treatment (40 µM) resulted in significant changes in 25% genes. A total of 15 common genes were altered by both MeHg and MPP(+), and dopaminergic signaling transduction was the most affected pathway. Proteomic analysis identified a total of 2496 proteins, of which 188, 233 and 395 proteins were differentially changed by 1 μM and 2.5 μM MeHg, and MPP(+) respectively. A total of 61 common proteins were changed by both MeHg and MPP(+) treatment. The changed proteins were mainly involved in energetic generation-related metabolism pathway (propanoate metabolism, pyruvate metabolism and fatty acid metabolism), oxidative phosphorylation, proteasome, PD and other neurodegenerative disorders. A total of 7 genes/proteins including Ube2l3 (Ubiquitin-conjugating enzyme E2 L3) and Th (Tyrosine 3-monooxygenase) were changed in both genomic and proteomic analysis. These results suggest that MeHg and MPP(+) share many similar signaling pathways leading to the pathogenesis of PD and other neurodegenerative diseases.

Abstract 2504: In-depth proteome quantification of hepatocellular carcinoma tissues reveals significant liver dysfunction

Abstract Hepatocellular carcinoma (HCC) is one of the most fatal cancers world-wide. Comparing with genome analyses, in-depth proteome quantification of HCC liver tissues is still inadequate. In this study, we combined both stable isotope labeling and multiple reaction monitoring label-free strategies for in-depth proteome quantification of both pooled and individual liver tissues with or without carcinoma. Among the 2,759 and 1,788 reliably quantified proteins by labeling and label-free methods, 266 significantly changed proteins showed consistent dysregulation trends in both pooled and individual tissues. We demonstrated that these proteins are widely involved in different biological processes of carcinoma progression. And many essential processes, such as protein synthesis and degradation, xenobiotic toxic substances and alcohol metabolisms and so on, are significantly dysregulated. For example, 6 members of eIF family, eukaryotic translation initiation factor 2/3/4/6 (EIF2S2, EIF3D, EIF3I, EIF3J, EIF4A1 and EIF6); 4 members of 40S ribosomal subunit, ribosomal protein 7/11/12/20 (RPS7, RPS11, RPS12 and RPS20); and 3 members of 60S ribosomal subunit, ribosomal protein L12/P0/P2 (RPL12, RPLP0 and RPLP2) are reliably quantified in our study, and all of them are significantly up-regulated in both pooled and individual proteome quantification, which implies that the protein synthesis is activated during the progress of HCC. Besides, various molecules for protein degradation, 2 members of Ubiquitin-like modifier activating enzyme, ie. E1 and E2; ubiquitin-like modifier activating enzyme 1 (UBA1) and ubiquitin-conjugating enzyme E2L 3 (UBE2L3); 10 members of proteasome, proteasome 26S subunit alpha type 4/5/7 (PSMA4, PSMA5 and PSMA7), beta type 2/9 (PSMB2 and PSMB9), ATPase 3 (PSMC3), non-ATPase 2/5/11 (PSMD2, PSMD5 and PSMD11), and proteasome activator subunit 1 (PSME1); and 10 members of heat shock proteins, heat shock 10 kDa protein (HSPE1), 27 kDa protein (HSPB1), DnaJ (Hsp40) homolog (DNAJB11), 60 kDa protein (HSPD1), 70 kDa protein 4/5/8/9 (HSPA4, HSPA5, HSPA8 and HSPA9), and 90 kDa proteins (HSP90AA1 and HSP90AB1) are all significantly up-regulated in both pooled and individual proteome quantification . Therefore, the protein degradation processes are also activated in HCC liver tissue The upstream regulators for the 266 significantly dysregulated proteins in both pooled and individual proteome quantification were analyzed. Finally, 15 and 9 upstream regulators were predicted in the activated and inhibited states, respectively. And the regulation trends of most of the proteins are consistent with the states of upstream regulators. 7 genes of the 15 activated up-stream regulators, MYC, MYCN, NFE2L2, CEBPB, HIF1A, SATB1 and XBP1, are transcription factors Note: This abstract was not presented at the meeting. Citation Format: Yexiong Tan, Fangjun Wang, Hanfa Zhou, Hongyang Wang. In-depth proteome quantification of hepatocellular carcinoma tissues reveals significant liver dysfunction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2504. doi:10.1158/1538-7445.AM2014-2504

Adaptive Expression of MicroRNA-125a in Adipose Tissue in Response to Obesity in Mice and Men

MicroRNAs are emerging as new mediators in the regulation of adipose tissue biology and the development of obesity. An important role of microRNA-125a has been suggested in the pathogenesis of insulin resistance (IR). Here, we characterized the function of microRNA-125a in adipose tissue in a context of experimentally-induced IR and obesity in mice and in obese patients. We showed time dependent overexpression of the microRNA in adipose tissue of BALB/c and C57BL/6J mice in response to high fat diet (HFD) feeding. MicroRNA-125a expression was downregulated in vitro in insulin resistant 3T3-L1 adipocytes and ex vivo in adipose tissue of obese patients. In vitro modulation of microRNA-125a expression in 3T3-L1 adipocytes did not affect glucose uptake. Gene set enrichment analysis (GSEA) identified significantly altered expression patterns of predicted microRNA-125a gene targets in transcriptomic datasets of adipose tissue from HFD-fed mice and obese patients. Among genes that contributed to global enrichment of altered expression of microRNA-125a targets, Thyrotroph embryonic factor (Tef), Mannan-binding lectin serine peptidase 1, Reticulon 2 and Ubiquitin-conjugating enzyme E2L3 were significantly differentially expressed in adipose tissue in these groups. We showed that Tef expression is reduced in adipose tissue of obese patients following gastric bypass surgery. Our findings indicate that microRNA-125a expression in adipose tissue adapts to IR and may play a role in the development of obesity in mice and obese subjects through uncoupled regulation of the expression of microRNA-125a and its targets.

A targeted association study in systemic lupus erythematosus identifies multiple susceptibility alleles

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.