Maf Proteins Research Articles

Abstract 2448 Functions of Small Maf Proteins in Senescence

Abstract 2448 Functions of Small Maf Proteins in Senescence

The Functional Significance of McMafF_G_K in Molluscs: Implications for Nrf2-Mediated Oxidative Stress Response

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal regulator of antioxidant gene expression in mammals, forming heterodimer complexes with small Maf proteins through its BZip domain. However, the underlying mechanism of Nrf2 action in molluscs remains poorly understood. The thick shell mussel, Mytilus coruscus, represents a model organism for the marine environment and molluscs interaction research. In this study, we used in silico cloning to obtain a small Maf homologue called McMafF_G_K from M. coruscus. McMafF_G_K possesses a typical BZip domain, suggesting its affiliation with the traditional small Maf family and its potential involvement in the Nrf2 signaling pathway. Transcriptional analysis revealed that McMafF_G_K exhibited a robust response to benzo[a]pyrene (Bap) in the digestive glands. However, this response was down-regulated upon interference with McMafF_G_K-siRNA. Interestingly, the expression levels of Nrf2, NAD(P)H: quinone oxidoreductase (NQO-1), and Glutathione Peroxidase (GPx), which are key players in oxidative stress response, showed a positive correlation with McMafF_G_K in digested adenocytes of M. coruscus. Furthermore, in vitro analysis of antioxidant capacity in digestive gland cells demonstrated that Bap exposure led to an increase in reactive oxygen species (ROS) levels, accompanied by an elevation in total antioxidant capacity (T-AOC), potentially counterbalancing the excessive ROS. Strikingly, transfection of McMafF_G_K siRNA resulted in a significant rise in ROS level and a down-regulation of T-AOC level. To validate the functional relevance of McMafF_G_K, a glutathione S-transferase (GST) pull-down assay confirmed its interaction with McNrf2, providing compelling evidence of their protein interaction. This study significantly contributes to our understanding of the functional role of McMafF_G_K in the Nrf2 signaling pathway and sheds light on its potential as a target for further research in oxidative stress response.

Regulation of Keap1-Nrf2 axis in temporal lobe epilepsy-hippocampal sclerosis patients may limit the seizure outcomes.

Accumulation of reactive oxygen species (ROS) exacerbates neuronal loss during seizure-induced excitotoxicity. Keap1 (Kelch-like ECH-associated protein1)-nuclear factor erythroid 2-related factor 2 (Nrf2) axis is one of the known active antioxidant response mechanisms. Our study focused on finding the factors influencing Keap1-Nrf2 axis regulation in temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) patients. Based on post-surgical follow-up data, patient samples (n = 26) were categorized into class 1 (completely seizure-free) and class 2 (only focal-aware seizures/auras), as suggested by International League Against Epilepsy (ILAE). For molecular analyses, double immunofluorescence assay and Western blot analysis were employed. A significant decrease in expression of Nrf2 (p < 0.005), HO-1; p < 0.02) and NADPH Quinone oxidoreductase1 (NQO1; p < 0.02) was observed in ILAE class 2. Keap1 (p < 0.02) and histone methyltransferases (HMTs) like SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) (p < 0.009) and enhancer of zeste homolog 2 (EZH2; p < 0.02) and methylated histones viz., H3K4me1 (p < 0.001), H3K9me3 (p < 0.001), and H3K27me3 (p < 0.001) was upregulated in ILAE class 2. Nrf2-interacting proteins viz., p21 (p < 0.001) and heat shock protein 90 (HSP90; p < 0.03) increased in class 1 compared to class 2 patients. Upregulation of HMTs and methylated histones can limit phase II antioxidant enzyme expression. Also, HSP90 and p21 that interfere with Keap1-Nrf2 interaction could contribute to a marginal increase in HO-1 and NQO1 expression despite histone methylation and Keap1. Based on our findings, we conclude that TLE-HS patients prone to seizure recurrence were found to have dysfunctional antioxidant response, in part, owing to Keap1-Nrf2 axis. The significance of Keap1-Nrf2 signaling mechanism in generation of phase II antioxidant response. Keap1-Nrf2 controls antioxidant response through regulation of phase II antioxidant enzymes like HO-1 (heme oxygenase-1), NQO1 (NADPH-Quinone Oxidoreductase1), and glutathione S-transferase (GST). Release of Nrf2 from negative regulation by Keap1 causes its translocation into nucleus, forming a complex with cAMP response-element binding protein (CBP) and small Maf proteins (sMaf). This complex subsequently binds antioxidant response element (ARE) and elicits and antioxidant response involving expression of phase II antioxidant enzymes. Reactive oxygen species (ROS) modify Cysteine 151 residue, p62 (sequsetosome-1), and interacts with Nrf2- binding site in Keap 1. p21 and HSP90 prevent Nrf2 interaction with Keap1. At transcriptional level, histone methyltransferases like EZH2 (enhancer of zeste homologue2), and SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) and corresponding histone targets viz., H3K27me3, H3K9me3, and H3K4me1 influence Nrf2 and Keap1 expression respectively.

Role of Activator Protein-1 Transcription Factor in Oral Cancer

Abstract: Cancer is a multi-step process involving alterations in epigenetic and genetic processes. Oral squamous cell carcinoma is a frequent oral malignancy that originates from the transformation of normal cells into malignant cells as a consequence of failures in a series of normal molecular and cellular processes. The mechanism of human carcinogenesis is often seen as a double-edged sword, with the body's system being thought to counteract the detrimental consequences of neoplastic cell proliferation while simultaneously promoting tumor development. Various transcription factors play a significant part in cancer regulation, with the activator protein-1 family of transcription factors (TFs) being the most prominent regulatory protein family. The Jun, Fos, ATF, and MAF protein families are all present in the AP-1 dimeric complex. While certain AP-1 proteins, including JunB and c-Fos, are known to be majorly oncogenic in function, experimental studies have shown that other AP-1 proteins, such as JunB and c-Fos, also play a critical role in tumor suppression. The aim of this review is to offer breakthrough information on the role of molecular mechanisms mediated by AP-1 TFs in tumor development and its environment.

Maf family transcription factors are required for nutrient uptake in the mouse neonatal gut.

There are fundamental differences in how neonatal and adult intestines absorb nutrients. In adults, macromolecules are broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely on internalization of whole macromolecules and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors MAFB and c-MAF as markers of terminally differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4α and HNF4γ, master regulators of enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of macromolecular nutrient uptake. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number, in response to loss of Maf proteins. Finally, we show that loss of BLIMP1, a repressor of adult enterocyte genes, shows highly overlapping changes in gene expression and similar defects in macromolecular uptake. This work defines transcriptional regulators that are necessary for nutrient uptake in neonatal enterocytes.

Structural basis of transcription regulation by CNC family transcription factor, Nrf2.

Several basic leucine zipper (bZIP) transcription factors have accessory motifs in their DNA-binding domains, such as the CNC motif of CNC family or the EHR motif of small Maf (sMaf) proteins. CNC family proteins heterodimerize with sMaf proteins to recognize CNC-sMaf binding DNA elements (CsMBEs) in competition with sMaf homodimers, but the functional role of the CNC motif remains elusive. In this study, we report the crystal structures of Nrf2/NFE2L2, a CNC family protein regulating anti-stress transcriptional responses, in a complex with MafG and CsMBE. The CNC motif restricts the conformations of crucial Arg residues in the basic region, which form extensive contact with the DNA backbone phosphates. Accordingly, the Nrf2-MafG heterodimer has approximately a 200-fold stronger affinity for CsMBE than canonical bZIP proteins, such as AP-1 proteins. The high DNA affinity of the CNC-sMaf heterodimer may allow it to compete with the sMaf homodimer on target genes without being perturbed by other low-affinity bZIP proteins with similar sequence specificity.

MafG-like contribute to copper and cadmium induced antioxidant response by regulating antioxidant enzyme in Procambarus clarkii

Avian musculoaponeurotic fibrosarcoma (Maf) proteins play an important role in Nrf2/Keap1 signaling pathway, which mainly resist the oxidant stress. The members of sMaf have a high homology basic leucine zipper (bZIP) and lack trans activation domain, and could interact with other transcriptional regulatory factors as a molecular chaperone. In this study, a full-length MafG-like gene was cloned from Procambarus Clarkii, designated as PcMafG-like, which consisted of an ORF length of 246 bp encoding 82 amino acids, a 5′ untranslated region (UTR) of 483 bp, and a 3′ UTR of 111 bp. The domain of PcMafG-like had a bZIP-Maf domain that binds to DNA. The cDNA sequence of PcMafG-like was 99 % similar to that of Penaeus vannamei. The mRNA of PcMafG-like was expressed in all tested tissues, and the highest expression was in muscle tissue. Under stimulation of Cu2+ and Cd2+, PcMafG-like was significantly up-regulated in hepatopancreas and gill, and the same result was testified by situ hybridization. The representative antioxidant genes, CAT, GPx and CZ-SOD, were significantly induced by Cu2+; CAT and GPx was induced by Cd2+. PcMafG-dsRNA significantly inhibited the expression of these up-regulated genes, but also inhibited the expression of other detected genes CZ-SOD, GST-θ and GST-1like. The antioxidant effect of PcMafG-like was further verified by oxidative stress markers (T-SOD, CuZnSOD, GPx, CAT, GSH and MDA) kits. Cu2+ and Cd2+ could induce the contents of these oxidative stress markers (MDA, GSH, CZ-SOD, CAT in Cu2+/Cd2+ treated group, and GSH-Px in Cd2+ group), while interference of PcMafG-like significantly inhibited the up-regulation. Furthermore, hematoxylin-eosin staining experiments showed that the degree of pathological damage was dose-dependent and time-dependent, and the pathological damage was more serious after dsRNA interfered with PcMafG-like. In addition, subcellular localization showed that PcMafG-like gene existed in nucleus. The recombinant protein PcMafG-like was expressed and purified in prokaryotic expression. The affinity analysis of promoter by agarose gel electrophoresis suggested that PcMafG-like could bind with CAT promoter in vitro. This indicated that PcMafG-like could activate antioxidant genes.

Deficiency of the bZIP transcription factors Mafg and Mafk causes misexpression of genes in distinct pathways and results in lens embryonic developmental defects.

Deficiency of the small Maf proteins Mafg and Mafk cause multiple defects, namely, progressive neuronal degeneration, cataract, thrombocytopenia and mid-gestational/perinatal lethality. Previous data shows Mafg −/−:Mafk +/- compound knockout (KO) mice exhibit cataracts age 4-months onward. Strikingly, Mafg −/−:Mafk −/− double KO mice develop lens defects significantly early in life, during embryogenesis, but the pathobiology of these defects is unknown, and is addressed here. At embryonic day (E)16.5, the epithelium of lens in Mafg −/−:Mafk −/− animals appears abnormally multilayered as demonstrated by E-cadherin and nuclear staining. Additionally, Mafg −/−:Mafk −/− lenses exhibit abnormal distribution of F-actin near the “fulcrum” region where epithelial cells undergo apical constriction prior to elongation and reorientation as early differentiating fiber cells. To identify the underlying molecular changes, we performed high-throughput RNA-sequencing of E16.5 Mafg −/−:Mafk −/− lenses and identified a cohort of differentially expressed genes that were further prioritized using stringent filtering criteria and validated by RT-qPCR. Several key factors associated with the cytoskeleton, cell cycle or extracellular matrix (e.g., Cdk1, Cdkn1c, Camsap1, Col3a1, Map3k12, Sipa1l1) were mis-expressed in Mafg −/−:Mafk −/− lenses. Further, the congenital cataract-linked extracellular matrix peroxidase Pxdn was significantly overexpressed in Mafg −/−:Mafk −/− lenses, which may cause abnormal cell morphology. These data also identified the ephrin signaling receptor Epha5 to be reduced in Mafg −/−:Mafk −/− lenses. This likely contributes to the Mafg −/−:Mafk −/− multilayered lens epithelium pathology, as loss of an ephrin ligand, Efna5 (ephrin-A5), causes similar lens defects. Together, these findings uncover a novel early function of Mafg and Mafk in lens development and identify their new downstream regulatory relationships with key cellular factors.

Target Gene Diversity of the Nrf1-MafG Transcription Factor Revealed by a Tethered Heterodimer.

Members of the cap'n'collar (CNC) family of transcription factors, including Nrf1 and Nrf2, heterodimerize with small Maf (sMaf) proteins (MafF, MafG, and MafK) and regulate target gene expression through CNC-sMaf-binding elements (CsMBEs). We recently developed a unique tethered dimer assessment system combined with small Maf triple-knockout fibroblasts, which enabled the characterization of specific CNC-sMaf heterodimer functions. In this study, we evaluated the molecular function of the tethered Nrf1-MafG (T-N1G) heterodimer. We found that T-N1G activates the expression of proteasome subunit genes, well-known Nrf1 target genes, and binds specifically to CsMBEs in the proximity of these genes. T-N1G was also found to activate genes involved in proteostasis-related pathways, including endoplasmic reticulum-associated degradation, chaperone, and ubiquitin-mediated degradation pathways, indicating that the Nrf1-MafG heterodimer regulates a wide range of proteostatic stress response genes. By taking advantage of this assessment system, we found that Nrf1 has the potential to activate canonical Nrf2 target cytoprotective genes when strongly induced. Our results also revealed that transposable SINE B2 repeats harbor CsMBEs with high frequency and contribute to the target gene diversity of CNC-sMaf transcription factors.

Nrf2 Regulation by Curcumin: Molecular Aspects for Therapeutic Prospects.

Nuclear factor erythroid 2 p45-related factor (2Nrf2) is an essential leucine zipper protein (bZIP) that is primarily located in the cytoplasm under physiological conditions. Nrf2 principally modulates endogenous defense in response to oxidative stress in the brain.In this regard, Nrf2 translocates into the nucleus and heterodimerizes with the tiny Maf or Jun proteins. It then attaches to certain DNA locations in the nucleus, such as electrophile response elements (EpRE) or antioxidant response elements (ARE), to start the transcription of cytoprotective genes. Many neoplasms have been shown to have over activated Nrf2, strongly suggesting that it is responsible for tumors with a poor prognosis. Exactly like curcumin, Zinc–curcumin Zn (II)–curc compound has been shown to induce Nrf2 activation. In the cancer cell lines analyzed, Zinc–curcumin Zn (II)–curc compound can also display anticancer effects via diverse molecular mechanisms, including markedly increasing heme oxygenase-1 (HO-1) p62/SQSTM1 and the Nrf2 protein levels along with its targets. It also strikingly decreases the levels of Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1) protein.As a result, the crosstalk between p62/SQSTM1 and Nrf2 could be used to improve cancer patient response to treatments. The interconnected anti-inflammatory and antioxidative properties of curcumin resulted from its modulatory effects on Nrf2 signaling pathway have been shown to improve insulin resistance. Curcumin exerts its anti-inflammatory impact through suppressing metabolic reactions and proteins such as Keap1 that provoke inflammation and oxidation. A rational amount of curcumin-activated antioxidant Nrf2 HO-1 and Nrf2-Keap1 pathways and upregulated the modifier subunit of glutamate-cysteine ligase involved in the production of the intracellular antioxidant glutathione. Enhanced expression of glutamate-cysteine ligase, a modifier subunit (GLCM), inhibited transcription of glutamate-cysteine ligase, a catalytic subunit (GCLC). A variety of in vivo, in vitro and clinical studies has been done so far to confirm the protective role of curcumin via Nrf2 regulation. This manuscript is designed to provide a comprehensive review on the molecular aspects of curcumin and its derivatives/analogs via regulation of Nrf2 regulation.

Loss of the MAF Transcription Factor in Laryngeal Squamous Cell Carcinoma.

MAF is a transcription factor that may act either as a tumor suppressor or as an oncogene, depending on cell type. We have shown previously that the overexpressed miR-1290 influences MAF protein levels in LSCC (laryngeal squamous cell carcinoma) cell lines. In this study, we shed further light on the interaction between miR-1290 and MAF, as well as on cellular MAF protein localization in LSCC. We confirmed the direct interaction between miR-1290 and MAF 3′UTR by a dual-luciferase reporter assay. In addition, we used immunohistochemistry staining to analyze MAF protein distribution and observed loss of MAF nuclear expression in 58% LSCC samples, of which 10% showed complete absence of MAF, compared to nuclear and cytoplasmatic expression in 100% normal mucosa. Using TCGA data, bisulfite pyrosequencing and CNV analysis, we excluded the possibility that loss-of-function mutations, promoter region DNA methylation or CNV are responsible for MAF loss in LSCC. Finally, we identified genes involved in the regulation of apoptosis harboring the MAF binding motif in their promoter region by applied FIMO and DAVID GO analysis. Our results highlight the role of miR-1290 in suppressing MAF expression in LSCC. Furthermore, MAF loss or mislocalization in FFPE LSCC tumor samples might suggest that MAF acts as a LSCC tumor suppressor by regulating apoptosis.

MAF and NF-κB Signaling Pathways Regulate Expression of APOBEC3B in Multiple Myeloma

IntroductionAPOBEC mutagenesis is a source of mutations in ~50% of human cancers, resulting from the over-expression of APOBEC proteins, including APOBEC3B (A3B). High expression of A3B is associated with poor prognosis and drug resistance in many cancers. In multiple myeloma, the chromosomal translocation t(14;16) leads to the over-expression of the transcription factor MAF through juxtaposition with the IGH super-enhancer. We have shown that in this poor prognosis sub-group A3B is also over-expressed and there is an enrichment for APOBEC signature mutations and a higher mutational load. It is not clear whether over-expression of A3B results from direct activation by MAF or indirectly through downstream pathways. In other cancers, mitosis, cell-cycle related gene expression, and the PKC/NF-κB signaling pathway may also affect expression of A3B.MethodsGene expression profile (GEP, U133 Plus 2.0) of 50 human myeloma cell lines (HMCLs) and 1310 myeloma patients was performed. mRNA levels of MAF and the AID /APOBEC family was investigated by qPCR in HMCLs. Western blotting and a DNA cytosine deaminase activity assay were used to detect A3B protein and analyze A3B enzymatic activity which results in mutagenesis, respectively. The effect of MAF on expression of A3B was investigated in inducible TRIPZ lentiviral shRNA c-MAF knockdown in HMCLs with high expression of MAF. Bortezomib, a proteasome inhibitor, and phorbol 12-myristate 13-acetate (PMA), a PKC dependent activator of the NF-κB signaling pathway, was used to investigate A3B expression in TRIPZ lentiviral shRNA MAF (MF KO) and A3B (A3B KO) knockdown HMCLs. Localization of A3B in regular HMCLs and cell lines with over-expression of A3B (generated using ViraPower T-Rex lentiviral expression system) was investigated by immunofluorescent analysis (IFA) using laser confocal and high resolution microscopy.ResultsBy microarray, high levels (log2>10) of A3B mRNA were found in approximately half of multiple myeloma patients. The majority of t(14;16) MAF patients had high levels of A3B. Increased expression of A3B was also detected in 72% of HMCLs. In t(14;16), MAF, and t(14;20), MAFB, HMCLs there was a strong positive correlation between MAF and A3B mRNA expression (r2= 0.51 and r2=0.43, respectively). On the basis of levels of MAF expression two groups of HMCLs were selected. Group 1 with high expression of MAF (MF) with the presence of a t(14;16) ARP1, CAG, JJN3, OCMY5, MM1S, and RPMI8226. Group 2 had low expression of MAF (non-MF) and was represented by cell lines with t(11;14) - U266, XG1 and KMS12BM; t(4;14) - KMS28PE and H929; or t(14;20) SACHI. We found a strong positive correlation (r2=0.68) between MAF and A3B mRNA expression in MF cell lines and no such correlation in the non-MF group (r2=0.09). Upon down-regulation of MAF by shRNA, JJN3 and RPMI8226 showed reduced expression of A3B protein (-33% and -29%, respectively) by 72 hours with accompanying reduction in cytosine deaminase activity (-23% and -26%, respectively). There was a positive correlation between expression of MAF protein and A3B protein in JJN3 MAF KO r2= 0.21, in RPMI8226 MAF KO r2=0.50; and a positive correlation between MAF protein and DNA cytosine deaminase activity in JJN3 MAF KO r2=0.21 and in RPMI8226 MAF KO r2=0.34. Bortezomib reduced expression of MAF and A3B and DNA cytosine deaminase activity in JJN3 and RPMI8226 cell lines. PMA increased the expression of A3B mRNA in HMCLs within 6 hours of treatment and lasted for 7 days after treatment. The highest sensitivity of A3B to PMA was detected in JJN3 and U266 cell lines, with increases of 4.4 and 8.2-fold, respectively, after 6 hours of treatment. Protein levels of A3B were increased 2.4-fold in both cell lines but DNA cytosine deaminase activity was increased (27%) only in JJN3 cells after PMA treatment.ConclusionsKnock down of MAF results in decreased expression and activity of A3B, indicating that MAF is a regulator of A3B expression in multiple myeloma. Expression of A3B in t(14;16) cases is dependent upon MAF expression but can also be increased through activation of the PKC/NF-κB signaling pathway, resulting in increased DNA damage. Understanding the mechanisms responsible for mutagenesis in the t(14;16) subgroup through A3B, which results in new clone formation and drug resistance in multiple myeloma will be highly beneficial in designing new therapeutic strategies. DisclosuresDavies:Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria.

MAFA Expression Promotes Proliferation of Myeloma Cells and Its Protein Stability Is Regulated By Proteasome Inhibitors

IntroductionMultiple myeloma (MM) patients with a t(14;16) or t(14;20) which result in high expression of MAF and MAFB, respectively, have a poor prognosis, and have not benefitted from the recent advances in MM therapy. Our previous work showed that post-translational modifications (PTM) further up-regulated MAF (Blood, 2016) and MAFB proteins (ASH Abstract #2091, 2014) conferring innate resistance to proteasome inhibitors (PI). The large MAF family of proteins contains a third member, MAFA. Little is known about its biological role in MM pathogenesis, as a probeset for MAFA is not present in gene expression profiling (GEP) analysis of molecular subgroups, and MAFA probes are not routinely used in fluorescence in situ hybridization (FISH) analysis. In the present study we therefore investigated the biological role of MAFA in MM and its effect on PI resistance.MethodsThe MAFA translocation in primary MM cells was determined by FISH analysis in 19 newly diagnosed MM patients. MAFA protein expression and protein level in response to PIs were assessed by immunoblotting and immunofluorescence staining (IFS). The effect of MAFA on proliferation of MM cells was measured by MTT assay. Annexin V staining and BrdU incorporation, analyzed by FACS analysis, was performed to determine the molecular action of MAFA on the survival of MM cells. MAFA expression was down-regulated in human MM cell lines (HMCLs). HMCLs using a lentiviral shRNA expression system. qRT-PCR analysis was used to determine MAFA mRNA expression in 32 HMCL and primary CD138+ plasma cells from 14 MM patients, and MAFA levels after shRNA treatment.ResultsFour levels of MAFA mRNA expression (relative to GAPDH) by RT-PCR analysis were determined: high levels were detected in 5 HMCLs, intermediate levels (100 to 500 ) in 10, low levels (10<100) in 7 and negative (<10) in 10 HMCLs. Among primary CD138+ patient cells (n=14), high levels were found in one patient, intermediate levels in 3, low levels in 4 and negative in 6 patients. FISH analysis found MAFA /t(8;14)(q24;q32) translocation in one out of 19 patients, which was also in the MF group by GEP analysis suggesting common downstream targets of MAFA with MAF and MAFB . MAFA protein was detected in the 6 HMCLs which expressed the highest MAFA mRNA levels by RT-PCR but not in the others.To determine if MAFA is required for MM cell survival, we knocked-down MAFA expression in three HMCLs that expressed high levels of MAFA . sh MAFA lentiviral supernatant infected HMCLs had 75% lower levels of MAFA mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, bioinformatically defined MAFA target genes were significantly decreased, including TLR4 (2.4-fold, P<0.01), NUAK1 (2.3-fold, p<0.01) and SPP1 (osteopontin, 2.2-fold, P<0.01), compared with HMCLs infected with control viral vector. Silencing MAFA expression significantly decreased the proliferation of HMCLs (62.5% decrease, P=2.1x10-4). Among 6 high MAFA expressing HMCLs, 3 co-expressed MAF and 2 co-expressed MAFB . To determine if MAFA synergized with MAF in promoting MM proliferation, we silenced MAFA and MAFB genes in cells co-expressing these genes. Simultaneously silencing MAFA and MAFB did not further decrease proliferation compared to either silencing MAFA or MAFB alone. These results indicate that MAFA promotes MM cell proliferation and that there is no synergetic effect between MAFA and MAFB in regulating MM proliferation.To determine if MAFA protein is regulated by PTM, we treated MM cells with PIs including bortezomib (Bzb) and carfilzomib (CFZ). Bzb induced stabilization of MAFA protein in a dose-dependent manner. Similar effects were seen with CFZ indicating that MAFA protein may be degraded by the ubiquitin proteasome system.ConclusionTaken together, our results indicate that increased levels of MAFA are seen in MM including the MAFA /t(8;14) translocation. High expression of MAFA mRNA and protein occurs in 18.7 % of MM cell lines. MAFA promotes myeloma proliferation and regulates TLR4, NUAK1 and SPP1 gene expression. The stability of MAF protein is regulated by PIs. These results provide new insights in understanding the role of the MAF family and, in particular, MAFA in MM pathogenesis. Experiments are ongoing to determine the function of MAFA in PI-resistance and to confirm MAFA targets in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup. DisclosuresMorgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria.

The small protein MafG plays a critical role in MC3T3-E1 cell apoptosis induced by simulated microgravity and radiation

Microgravity and radiation exposure-induced bone damage is one of the most significant alterations in astronauts after long-term spaceflight. However, the underlying mechanism is still largely unknown. Recent ground-based simulation studies have suggested that this impairment is likely mediated by increased production of reactive oxygen species (ROS) during spaceflight. The small Maf protein MafG is a basic-region leucine zipper-type transcription factor, and it globally contributes to regulation of antioxidant and metabolic networks. Our research investigated the role of MafG in the process of apoptosis induced by simulated microgravity and radiation in MC3T3-E1 cells. We found that simulated microgravity or radiation alone decreased MafG expression and elevated apoptosis in MC3T3-E1 cells, and combined simulated microgravity and radiation treatment aggravated apoptosis. Meanwhile, under normal conditions, increased ROS levels facilitated apoptosis and downregulated the expression of MafG in MC3T3-E1 cells. Overexpression of MafG decreased apoptosis induced by simulated microgravity and radiation. These findings provide new insight into the mechanism of bone damage induced by microgravity and radiation during space flight.

Epigallocatechin-3-gallate Alleviates Vanadium-Induced Reduction of Antioxidant Capacity via Keap1-Nrf2-sMaf Pathway in the Liver, Kidney, and Ovary of Laying Hens.

This study evaluated the effect of epigallocatechin-3-gallate (EGCG) alleviating the reduction of antioxidant capacity induced by dietary vanadium (V) in the liver, kidney, and ovary of laying hens. Furthermore, Kelch-like ECH-associated protein 1(Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-small Maf proteins (sMaf) pathway was explored to reveal the molecular mechanism. A total of 768 40-week-old Hyline-Brown laying hens were randomly allocated to 4 groups with 8 pens per group and 24 hens per pen. The experimental groups were as follows: control (basal diet); V15, control + 15 mg/kg V; EGCG150, control + 150 mg/kg EGCG; V15 + EGCG150, V15 + 150 mg/kg EGCG. Our results revealed that dietary EGCG supplementation completely alleviated the V-induced reductions of hen-day egg production, average egg weight, Haugh unit, albumen height, eggshell strength, and eggshell thickness. Dietary EGCG supplementation completely prevented the V-induced reductions of serum follicle-stimulating hormone and luteinizing hormone levels. Besides, dietary EGCG supplementation reversed the V-induced increments of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr), and uric acid (UA). In addition, dietary EGCG supplementation partially alleviated the V-induced reductions of the enzyme activities and gene expressions of superoxidative dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px). Furthermore, dietary EGCG supplementation partially alleviated the V-induced reductions of Nrf2 and sMaf gene expressions, and the increments of Keap1 gene expression. In summary, EGCG partially alleviated V-induced reduction of antioxidant capacity through Keap1-Nrf2-sMaf pathway in the liver, kidney, and ovary of laying hens.

Novel serine/threonine-O-glycosylation with N-acetylneuraminic acid and 3-deoxy-D-manno-octulosonic acid by bacterial flagellin glycosyltransferases.

Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.

Aeromonas caviae motility and glycosylation

Aeromonas are Gram-negative facultative anaerobic rods, which inhabit various aquatic environments and are pathogens of both warm and cold-blooded animals. In humans they cause gastro-enteritis and wound infections. They are motile in liquid environments by a single polar type of flagellum. The flagellum plays an important role for the bacterial colonisation and the adhesion to the host cells. The Aeromonaspolar flagella filament is a polymer composed of two flagellins, FlaA and FlaB. The flagellins are O-linked glycosylated through the addition of the unusual bacterial sugar pseudaminic acid to serine and threonine residues within the flagellins D2/D3 domain. The addition of this sugar is essential for flagella filament assembly and bacterial motility. The flagellin’s are modified by between 6 – 8 sugar residues that occupy the potential 14 sites of attachment. Motility accessory factors (Maf proteins) are candidate enzymes for transferring glycan molecules to the flagellin (glycosyltransferases transferring sugar to flagellin) due to their genetic location and motility phenotypes associated with disruption mutants. This study utilised site-directed mutagenesis to change the potential sites of flagellin glycosylation to assess the effect of these mutations on motility by swimming assays and flagella filament formation by electron microscopy. The analysis of different numbers of site-directed mutants suggest that some sites are more important than others and that the removal of 4 sites results in greatly reduced motility.

Structural Characterization of the Covalent Binding of Keap1 and Thiol‐containing Small Molecules

Oxidative stress is involved in a wide range of human diseases such as cancer, diabetes, and Alzheimer’s disease. High levels of reactive oxygen species (ROS) cause oxidative damage to DNA and other molecules vital to cell function, such as membrane lipids and proteins. Kelch‐like ECH‐associated protein 1 (Keap1) is a primary regulator of the body’s natural defense system against oxidative stress. In the absence of oxidative stress, Keap1 forms a homodimer that binds a single molecule of transcription factor nuclear factor‐like 2 (Nrf2), causing Nrf2 ubiquitination and degradation. Oxidative stress causes Keap1 to release Nrf2, allowing it to translocate to the nucleus, bind to Maf protein, and activate the production of antioxidant enzymes by binding to the antioxidant response element (ARE). Blocking the protein‐protein interaction (PPI) between Keap1 and Nrf2 could have great therapeutic value. The antioxidant enzymes upregulated by free Nrf2 provide an efficient, catalytic detoxification of ROS. However, as PPIs frequently take place between large, flat interfaces on the two participating proteins, these interactions can be difficult to block. A previous study by our collaborators at UCSF identified 43 thiol‐containing compounds (monophores) that can be covalently tethered to Keap1 at a cysteine near the protein interface. The goal of this project is to characterize the interaction between Keap1 and the monophores using X‐ray crystallography to obtain structures of the protein‐monophore complexes, or adducts. This structural data will allow us to judge which compounds are suitable for scaffold optimization, a technique in which a larger molecule is built from an initial smaller compound to improve its potency and selectivity. Information about the binding modes of the monophores could also be incorporated into the design of other types of drugs that have proven effective in blocking PPIs, such as macrocycles. To identify adduct crystallization conditions, a DTNB assay, which measures the number of reactive sulfhydryl groups in a protein, was used to screen buffer conditions without dithiothreitol (DTT) present. A high‐throughput version of this assay was developed and utilized, in combination with differential scanning fluorimetry (DSF), to identify stabilizing conditions in which adduct can form. This assay was validated with a monophore known to have a stabilizing effect on Keap1, and a new buffer condition (50 mM EPPS, 50 mM NaCl, pH 8.0) was identified in which adduct crystallization attempts can be made.Support or Funding InformationThis work was supported by the Arnold and Mabel Beckman Foundation.

Effect of Triptolide on Cardiac Function in Arthritic Rats and Its Mechanism

To observe the changes of cardiac function in arthritic rats and the effect of triptolide on it. Forty rats were divided in random into normal control (NC) group, model control (MC) group, leflunomide (LEF) group and triptolide (TP) group. Except for the normal group, rats in the other three groups were injected with Freund's complete adjuvant to create arthritic inflammation in the right hind paws, and the interventional drug was administered on the 12th day after the inflammation. By treating for 30 d, the cardiac function of rats was detected by left ventricular catheterization. The expressions of superoxide dismutase (SOD), malondialdehyde (MDA), reacitve oxygen species (ROS), total antioxidation (T-AOC), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) in serum were measured by enzyme-linked immunosorbent assay. The expressions of keap-like protein 1 ( Keap1), muscular aponeurotic fibrosarcom ( maf) and nuclear factor-E2 related factor2 ( Nrf2) mRNAs in cardiac tissue were detected by real-time PCR. The expressions of Keap1, maf and Nrf2 proteins in heart tissues were detected by Western blot. Comparing with the normal group, the heart rate (HR), heart index (HI), left ventricular systolic pressure (LVSP), and left ventricular end-diastolic pressure (LVEDP) of the model group were significantly increased, whereas the maximum change rate of ventricular pressure rise or decline (±dp/dtmax) was significantly decreased ( P<0.01). SOD, MDA, ROS, T-AOC, and TNF-α were all increased, and IL-10 was significantly decreased ( P<0.01). The mRNA and protein expressions of Keap1, maf and Nrf2 in heart tissues were increased ( P<0.01). Comparing with the model group, HR, HI, LVSP, and LVEDP in the triptolide group were significantly decreased, whereas the ±dp/dtmax was significantly increased ( P<0.01). SOD, MDA, T-AOC, ROS, TNF-α decreased while the IL-10 increased ( P<0.05, P<0.01). The expressions of Keap1, maf and Nrf2 mRNAs and proteins in the heart tissues of the triptolide group were decreased ( P<0.01). Triptolide could improve cardiac function in arthritic rats, and the mechanism may related to its ability of improving the anti-oxidationin cardiomyocytes, reducing oxidative stress damage, and inhibiting abnormal immune inflammatory response.

The deubiquitinase USP7 stabilizes Maf proteins to promote myeloma cell survival

The Maf proteins, including c-Maf, MafA, and MafB, are critical transcription factors in myelomagenesis. Previous studies demonstrated that Maf proteins are processed by the ubiquitin-proteasome pathway, but the mechanisms remain elusive. This study applied MS to identify MafB ubiquitination-associated proteins and found that the ubiquitin-specific protease USP7 was present in the MafB interactome. Moreover, USP7 also interacted with c-Maf and MafA and blocked their polyubiquitination and degradation. Consistently, knockdown of USP7 resulted in Maf protein degradation along with increased polyubiquitination levels. The action of USP7 thus promoted Maf transcriptional activity as evidenced by luciferase assays and by the up-regulation of the expression of Maf-modulated genes. Furthermore, USP7 was up-regulated in myeloma cells, and it was negatively associated with the survival of myeloma patients. USP7 promoted myeloma cell survival, and when it was inhibited by its specific inhibitor P5091, myeloma cell lines underwent apoptosis. These results therefore demonstrated that USP7 is a deubiquitinase of Maf proteins and promotes MM cell survival in association with Maf stability. Given the significance of USP7 and Maf proteins in myeloma genesis, targeting the USP7/Maf axle is a potential strategy to the precision therapy of MM.