C-terminal Deletion Research Articles

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin

SummaryThe C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.

Role of α-Synuclein Regions in Nucleation and Elongation of Amyloid Fiber Assembly.

α-Synuclein is an intrinsically disordered protein whose aggregation in the form of amyloid fibers is directly implicated in Parkinson's disease and other neurological disorders. α-Synuclein is composed of three different regions. The central region (61-95), called NAC, is responsible for protein fibrillation. The N-terminal region (1-61) has some helical propensity and can be divided into H1 (1-31) and H2 (32-61), while the highly acidic C-terminal region (96-140) is completely disordered. It has been postulated that the acidic character of the C-terminus, as well as the interaction between the soluble N- and C- terminal parts, protects the NAC region from fibrillation. In consequence, N- and C-terminal deletions increase α-synuclein fibrillation. Both N- and C-terminal truncations are common in synucleinopathies, but despite their clinical relevance, to date, there are no systematic and exhaustive studies that quantify the effect of these truncations in fiber nucleation and elongation. In this work, we measured both nucleation and fibrillation elongation kinetics in order to study the influence of N- and C-terminal deletions, including the simultaneous deletion of several regions, in α-synuclein fibrillation. We also tested whether the fibrillation prone mutation A53T had an additional effect when combined with truncations. Furthermore, our cross-seeding experiments showed that the deletions studied induce changes in fiber morphology. Our results unravel then the role of the different α-synuclein regions and the A53T mutation in the nucleation and elongation of amyloid fibers.

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation.

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

Identification of the Epitope Recognized by the Anti-Red Sea Bream Iridovirus (RSIV) Monoclonal Antibody M10 Using a Phage Display RSIV Peptide Library

Red sea bream iridoviral disease (RSIVD) spreads readily throughout the marine environment because of its wide variety of host species. Use of a rapid diagnostic method, such as the immunofluorescence antibody test, helps to control the disease, so the anti-RSIV monoclonal antibody M10 (mAb M10) has been developed in Japan. In the present study, we carried out epitope mapping using the phage display method to identify the antigen of mAb M10. A phage display RSIV peptide library was constructed to cover the entire genome of RSIV, then phage clones recognized by the antibody were selected by biopanning. The selected clones harbored partial fragments of the laminin-type epidermal growth factor-like domain (LEGFD) gene. N-terminal and C-terminal deletion peptides were then prepared from the amino acid sequence deduced from the smallest fragment to precisely determine the epitope. Finally, seven amino acids, EYDCPEY, located in the extracellular domain of the LEGFD protein were determined to be the epitope. Identical residues of the epitope were also identified from the LEGFD protein in other megalocytiviruses including the infectious spleen and kidney necrosis virus and turbot reddish body iridovirus. mAb M10 is considered to be widely available for the diagnostics of megalocytivirus infections.

Identification of sequence-specific interactions of the CD44-intracellular domain with RUNX2 in the transcription of matrix metalloprotease-9 in human prostate cancer cells.

Aim: The Cluster of differentiation 44 (CD44) transmembrane protein is cleaved by γ-secretase, the inhibition of which blocks CD44 cleavage. This study aimed to determine the biological consequence of CD44 cleavage and its potential interaction with Runt-related transcription factor (RUNX2) in a sequence-specific manner in PC3 prostate cancer cells.Methods: Using full-length and C-terminal deletion constructs of CD44-ICD (D1-D5) expressed as stable green fluorescent protein-fusion proteins in PC3 cells, we located possible RUNX2-binding sequences.Results: Chromatin immunoprecipitation assays demonstrated that the C-terminal amino acid residues between amino acids 671 and 706 in D1 to D3 constructs were indispensable for sequence-specific binding of RUNX2. This binding was minimal for sequences in the D4 and D5 constructs. Correspondingly, an increase in matrix metalloprotease-9 (MMP-9) expression was observed at the mRNA and protein levels in PC3 cells stably expressing D1–D3 constructs.Conclusion: These results provide biochemical evidence for the possible sequence-specific CD44-ICD/RUNX2 interaction and its functional relationship to MMP-9 transcription in the promoter region.

Drosophila Mon1 and Rab7 interact to regulate glutamate receptor levels at the neuromuscular junction.

Regulation of post-synaptic receptors plays an important role in determining synaptic strength and plasticity. The Drosophila larval neuromuscular junction (nmj) has been used extensively as a model to understand some of these processes. In this context, we are interested in the role of Drosophila Monensin sensitivity protein 1 (DMon1) in regulating glutamate receptor (GluRIIA) levels at the nmj. DMon1 is an evolutionarily conserved protein which, in complex with calcium caffeine zinc sensitivity1 (CCZ1), regulates the conversion of early endosomes to late endosomes through recruitment of Rab7. C-terminal deletion mutants of Dmon1 (Dmon1Δ181) exhibit lethality. The escapers have a short life span and exhibit severe motor defects. At the nmj, these mutants show defects in synaptic morphology and a strong increase in GluRIIA levels. The mechanism by which Dmon1 regulates GluRIIA is unclear. In this study, we have characterized an EMS mutant referred to as pog1 and demonstrate it to be an allele of Dmon1. Further, we have examined the role of rab7 in regulating GluRIIA. We show that similar to Dmon1, knock-down of rab7 using RNAi in neurons, but not muscles, leads to an increase in GluRIIA. Loss of one copy each of Dmon1 and rab7 leads to a synergistic increase in receptor expression. Further, overexpression of an activated Rab7 can rescue the GluRIIA phenotype observed in Dmon1 Δ181 mutants. Together, these results highlight a neuronal role for Rab7 in GluRIIA regulation and underscore the importance of the endo-lysosomal pathway in this process.

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An invitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata

Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

C-terminal sequence deletion effect on antioxidative characteristics of VLPVPQK bioactive peptide from buffalo milk casein

In the recent time, demand for nontoxic antioxidative substances such as peptides has boomed among pharmacologists. In the present investigation, peptides, VLPVPQ and VLPVP were generated from parent antioxidative peptide, VLPVPQK. Initially, all these peptides were studied in-silico for biophysical properties like hydrophobicity, structure, Extinction coefficient, isoelectric point, grand average hydropathy value and Wimley White whole residue hydrophobicity. Inspite of this, homology relationship among organisms, taxonomy, digestibility assessment for proteases, solubility, melting temperature index, half-life time in-vitro and in-vivo, instability index and aliphatic index was compared in-silico. Further, based on these properties, VLPVP was compared with parent peptide, VLPVPQK for antioxidative potential. VLPVP exhibited lesser antioxidative potential by chemical method and in-vitro hydrogen peroxide induced oxidative stress conditions generated in fibroblast cells compared to VLPVPQK. The proliferative potential, cell injury marker, nitric oxide production and pro-inflammatory, Tumor Necrosis Factor-α was measured to compare cytoprotective antioxidative potential. The results displayed the cytoprotective potential of VLPVPQK. In addition, the present study suggested that glutamine and lysine are responsible for the c-terminal sequence based response and cytoprotective potential of milk bioactive peptide, VLPVPQK. Other than this, hydrophobic amino acids and positive charge of the VLPVPQK is also responsible for the cytoprotective potential.

Deciphering the molecular mechanisms underlying the plasma membrane targeting of PRMT8

Arginine methylation plays crucial roles in many cellular functions including signal transduction, RNA transcription, and regulation of gene expression. Protein arginine methyl-transferase 8 (PRMT8), a unique brain-specific protein, is localized to the plasma membrane. However, the detailed molecular mechanisms underlying PRMT8 plasma membrane targeting remain unclear. Here, we demonstrate that the N-terminal 20 amino acids of PRMT8 are sufficient for plasma membrane localization and that oligomerization enhances membrane localization. The basic amino acids, combined with myristoylation within the N-terminal 20 amino acids of PRMT8, are critical for plasma membrane targeting. We also found that substituting Gly-2 with Ala [PRMT8(G2A)] or Cys-9 with Ser [PRMT8(C9S)] induces the formation of punctate structures in the cytosol or patch-like plasma membrane localization, respectively. Impairment of PRMT8 oligomerization/dimerization by C-terminal deletion induces PRMT8 mis-localization to the mitochondria, prevents the formation of punctate structures by PRMT8(G2A), and inhibits PRMT8(C9S) patch-like plasma membrane localization. Overall, these results suggest that oligomerization/dimerization plays several roles in inducing the efficient and specific plasma membrane localization of PRMT8.

Importance of C-Terminal Extension in Thermophilic 1,4-α-Glucan Branching Enzyme from Geobacillus thermoglucosidans STB02.

By sequence comparison, the majority of 1,4-α-glucan-branching enzymes (GBEs) consist of an N-terminal carbohydrate-binding domain, a TIM-barrel catalytic domain, and a C-terminal all-beta domain. Among these structures, the GBE from Geobacillus thermoglucosidans STB02 uniquely has a highly charged 26-amino-acid C-terminal extension, whose functional roles are the least understood. In this research, the functional significance of the C-terminal domain in GBE from G. thermoglucosidans STB02 and its extension were assessed using a C-terminal deletion analysis. Mutants lacking of more than 7 residues of the C-terminal all-beta domain could not be detected in lysates of their Escherichia coli expression strains, suggesting that an intact all-beta domain is required for structural stability. In contrast, truncation of the C-terminal extension resulted in greater stability and solubility than the wild type, as well as a lower sensitivity to the presence of added metal ions. Comparison of this mutant with the wild type suggests that the interaction of metal ions with the C-terminal extension influences performance of this enzyme.

Identification and functional characterization of the transcription factor NF-κB subunit p65 in common carp (Cyprinus carpio L.)

p65 is an important subunit of the transcription factor NF-κB in the regulation of immune response. In the present study, the p65 cDNA was identified from common carp (Cyprinus carpio L.) (named Ccp65). Phylogenetic analysis revealed that Ccp65 located in the same clade as piscine p65 and exhibited closest relationship to that of Ctenopharyngodon idella. Ccp65 was constitutively expressed in all the examined tissues. Aeromonas hydrophila and poly(I:C) can induce the expression of Ccp65 in the designated tissues and the Ccp65 expression was up-regulated in HKLs following LPS and poly(I:C) stimulation. In addition, the nuclear localization signal (NLS) and C-terminal domain are the important elements of Ccp65. Immunofluorescence assay revealed that the nuclear localization signal deletion mutation of Ccp65 (Ccp65ΔNLS) failed to translocate to the nucleus even though stimulation with poly(I:C) or LPS, and the C-terminal domain deletion mutation of Ccp65 (Ccp65ΔC) did not up-regulate the luciferase activity. Furthermore, Ccp65 can induce the expression of il-1β and tnf-α. And LPS and poly(I:C) inducing the expression of il-1β and tnf-α, is dependent on the Ccp65. Taken altogether, these findings lay the foundations for future research to investigate the mechanisms underlying fish p65.

P11. Abstract Title: Investigating the resistance of ADAMTS13 towards Alpha 2-Macroglobulin

<h3>Background</h3> ADAMTS13 is a metalloprotease that regulates the platelet-capturing capacity of von Willebrand factor. ADAMTS13 is secreted in its active form and has a circulating half-life of 4-7 days despite the presence of relatively high concentrations of inhibitors known to target other members of the ADAMTS protease family. This suggests that ADAMTS13 is resistant to inhibition by natural protease inhibitors, such as Alpha 2-Macroglobin (A2M) and tissue inhibitors of metalloproteinases (TIMPs). ADAMTS13 is known to adopt a closed conformation whereby C-terminal CUB domains bind to the proximal SPACER domain. The impact of this closed form of ADAMTS13 on accessibilty of the active site is not currently known. <h3>Methods</h3> To better understand the mechanism of ADAMTS13 resistance to natural protease inhibitors, C-terminal deletion constructs of ADAMTS13 and chimeras between ADAMTS13/ADAMTS5 were generated. Resistance was tested by examining the activity of ADAMTS13 variants in the presence of A2M using FRETS-VWF73; and then confirmed by Western blot examining the presence of the cleaved form of A2M. <h3>Results</h3> Activity assay showed that both full length (FL) -ADAMTS13 and MDTCS were resistant towards A2M up to 24 hours. Further, FL-ADAMTS13 and MDTCS were unable to cleave A2M; and therefore, were not inhibited. The MD variant in the presence of A2M showed marked reduction in activity and cleaved A2M. Therefore, MD was inhibited by A2M. <h3>On-going Studies</h3> Additional studies with ADAMTS5 (MD5TCS13, MD5TCST1-CUB2(13) and MD13TCS5) are on-going and should shed additional light on this unique property of ADAMTS13. These experiments will be repeated using the 4 isoforms of TIMPs; which are direct inhibitors of the metalloprotease active site. <h3>Conclusion</h3> Based on our domain truncations of ADAMTS13, its resistance towards A2M is likely mediated by proximal domains closest to its active site. On-going and future studies will try to determine the exact mechanism of resistance.

P5377A SUMO1-SIM motif within the C-terminal alpha-helical domain of S100A1 prevents its proteasomal degradation in cardiomyocytes

Abstract S100A1, a EF-hand calcium (Ca2+) sensor protein, predominantly expressed in cardiomyocytes, improves contractile performance and energy metabolism targeting activity of downstream key factors such as SERCA2a and F1ATPase. Vice versa, decreased cardiac S100A1 expression in heart failure thereby accelerates the progression to contractile and energetic failure. Although previous studies showed that transcriptional inhibition may contribute to the loss of S100A1 expression, little is known with respect to its post-translational stability in cardiomyocytes. As such, advanced understanding of the molecular mechanisms that may regulate S100A1 degradation may be of interest to target its stability in the failing heart. In search of protein stabilizing motifs, we identified a Small Ubiquitin-related Modifier1 (SUMO1) interaction motif (SIM) in the S100A1 C-terminal domain extending from AA 69–87 as well as multiple ubiquitylatable Lysin-residues within the N-terminal domain by an in silico structural analysis. Given the role of the C-terminal domain of S100A1 in accommodating target binding and activity modulation, we hypothesized that these residues may convey a dual function and also contribute to S100A1 protein stability. To answer this question, we generated different Myc-tagged and non-tagged C-terminal deletion mutants (Myc-S100a1ΔCTs/S100a1ΔCTs) within the SIM motif encompassing AA 1–74 and AA 1–83 besides the full length AA 1–94 (S100A1 -FL). Constructs packaged into bicistronic recombinant Adenovirus Serotype 5 also contained GFP to control for sufficient transduction. Ad5-mediated gene delivery of S100a1-Fl to cell culture resulted in robust S100A1-FL protein expression. In contrast, delivery of the truncated S100a1 mutants did not yield detectable S100A1ΔCT protein variants, whereas the presence of mRNA from all S100a1ΔCT variants by PCR excluded transcriptional inhibition or blockade as a potential cause for the failure to detect S100A1ΔCT protein variants. When a proteasomal inhibitor (MG-132) was then used, S100A1ΔCT variants were detected at protein level in tested models indicating that proteasomal degradation may target SIM deleted S100A1 ΔCT variants. This notion was corroborated by unchanged expression levels of FL-S100A1 as well as deletion C-terminal mutants containing the entire SIM motif. Our novel results indicate that the C-terminal domain of S100A1 may contain a SIM motif which shields S100A1 protein against proteasomal degradation. SUMOylation analysis as well as site-directed mutagenesis of the motif is subsequently required to determine conditions where S100A1 SUMOylation may be decreased (i.e. heart failure) and which specific AA residues may convey this action. Overall, the S100A1 C-terminus may play a role in target recognition as well as in the regulation of S100A1's protein stability. In vivo studies will be required to provide the ultimate proof for this novel hypothesis. Acknowledgement/Funding DZHK

UBAP1 mutations cause juvenile-onset hereditary spastic paraplegias (SPG80) and impair UBAP1 targeting to endosomes.

We aimed to find a new causative gene and elucidate the molecular mechanisms underlying a new type of hereditary spastic paraplegia (HSP). Patients with HSP were recruited from the Japan Spastic Paraplegia Research Consortium (JASPAC). Exome sequencing of genomic DNA from patients in four families was carried out, followed by Sanger sequencing of the UBAP1 gene. A mouse homolog of one UBAP1 frameshift mutation carried by one of the patients was created as a disease model. Functional properties of the UBAP1 wild type and UBAP1-mutant in mouse hippocampus neurons were examined. We identified three novel heterozygous loss of function mutations (c.425_426delAG, c.312delC, and c.535G>T) in the UBAP1 gene as the genetic cause of a new type of HSP (SPG80). All the patients presented identical clinical features of a pure type of juvenile-onset HSP. Functional studies on mouse hippocampal neurons revealed that the C-terminal deletion UBAP1-mutant of our disease model had lost its ability to bind ubiquitin in vitro. Overexpression of the UBAP1 wild type interacts directly with ubiquitin on enlarged endosomes, while the UBAP1-mutant cannot be recruited to endosome membranes. Our study demonstrated that mutations in the UBAP1 gene cause a new type of HSP and elucidated its pathogenesis. The full-length UBAP1 protein is involved in endosomal dynamics in neurons, while loss of UBAP1 function may perturb endosomal fusion and sorting of ubiquitinated cargos. These effects could be more prominent in neurons, thereby giving rise to the phenotype of a neurodegenerative disease such as HSP.

Methylosome protein 50 associates with the purinergic receptor P2X5 and is involved in osteoclast maturation.

Purinergic signaling plays important roles in bone. P2X5, a member of ligand-gated ion channel receptors, has been demonstrated to regulate osteoclast maturation. However, the molecular mechanism of P2X5-mediated osteoclast regulation remains unclear. Here, we identified methylosome protein 50 (MEP50), a critical cofactor of the protein arginine methyltransferase 5 (PRMT5), as a P2X5-associating molecule. RNAi-mediated knockdown of MEP50 results in decreased formation of mature osteoclasts. MEP50 associates with P2X5, and this association requires the C-terminal intracellular region of P2X5. Additionally, impaired maturation of P2X5-deficient osteoclasts could be restored by transduction of full-length P2X5, but not a C-terminal deletion mutant of P2X5. These results indicate that P2X5 associates with MEP50 and suggest a link between the PRMT5 complex and P2X5 signaling in osteoclast maturation.

Amino and carboxy-terminal extensions of yeast mitochondrial DNA polymerase assemble both the polymerization and exonuclease active sites

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection.

The role of the tight-junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C-terminal cytosolic domain in HCV infection. We expressed a series of C-terminal deletion mutants in Huh-7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN-C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans-Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN-C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN-C18. However, in the context of full-length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN-C18 in human-polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.

An insight into the folding and stability of Arabidopsis thaliana SOG1 transcription factor under salinity stress in vitro

The present study describes the biophysical characterization of Arabidopsis thaliana SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) protein, a NAC domain transcription factor which plays central role in DNA damage response pathway, under salinity stress in vitro. Tryptophan fluorescence studies using purified recombinant wild type (full length) AtSOG1 and its N-terminal or C-terminal deletion forms (AtSOG1ΔNAC and AtSOG1ΔCT respectively) have revealed high salinity induced conformational change due to removal of the N-terminal NAC domain. Bis-ANS binding assays indicate that removal of the N-terminal NAC domain increases the surface hydrophobic binding sites, while the C-terminal region of SOG1 also plays important role in regulating the surface hydrophobicity aspects following exposure to high salinity. Circular dichroism (CD) spectral studies have indicated that removal of the N-terminal NAC domain affects the structural conformation of the protein under high salt concentration. Urea-induced equilibrium unfolding studies revealed decreased stability of C-terminal region due to removal of the N-terminal NAC domain. In vitro aggregation studies have indicated higher propensity of aggregation of AtSOG1ΔNAC due to salt treatment. Overall, our results provide evidence for the importance of both N-terminal NAC domain and the C-terminal region in regulating the stability of SOG1 protein under salinity stress in vitro.

NDRG2 Sensitizes Myeloid Leukemia to Arsenic Trioxide via GSK3β-NDRG2-PP2A Complex Formation.

N-Myc downstream-regulated gene 2 (NDRG2) was characterized as a tumor suppressor, inducing anti-metastatic and anti-proliferative effects in several tumor cells. However, NDRG2 functions on anticancer drug sensitivity, and its molecular mechanisms are yet to be fully investigated. In this study, we investigated the mechanism of NDRG2-induced sensitization to As2O3 in the U937 cell line, which is one of the most frequently used cells in the field of resistance to As2O3. NDRG2-overexpressing U937 cells (U937-NDRG2) showed a higher sensitivity to As2O3 than mock control U937 cell (U937-Mock). The higher sensitivity to As2O3 in U937-NDRG2 was associated with Mcl-1 degradation through glycogen synthase kinase 3β (GSK3β) activation. Inhibitory phosphorylation of GSK3β was significantly reduced in U937-NDRG2, and the reduction was diminished by okadaic acid, a protein phosphatase inhibitor. NDRG2 mediated the interaction between GSK3β and protein phosphatase 2A (PP2A), inducing dephosphorylation of GSK3β at S9 by PP2A. Although the C-terminal deletion mutant of NDRG2 (ΔC NDRG2), which could not interact with PP2A, interacted with GSK3β, the mutant failed to dephosphorylate GSK3β at S9 and increased sensitivity to As2O3. Our findings suggest that NDRG2 is a kind of adaptor protein mediating the interaction between GSK3β and PP2A, inducing GSK3β activation through dephosphorylation at S9 by PP2A, which increases sensitivity to As2O3 in U937 cells.