Proteomic Analysis Identifies Ribosome Reduction as an Effective Proteotoxic Stress Response [Cell Biology]

October 21st, 2015 by

Stress responses are adaptive cellular programs that identify and mitigate potentially dangerous threats. Misfolded proteins are a ubiquitous and clinically relevant stress. Trivalent metalloids, such as arsenic, have been proposed to cause protein misfolding. Using tandem mass tag-based mass spectrometry, we show that trivalent arsenic results in widespread reorganization of the cell from an anabolic to a catabolic state. Both major pathways of protein degradation, the proteasome and autophagy, show increased abundance of pathway components, increased functional output, and are required for survival. Remarkably, cells also showed a downregulation of ribosomes at the protein level. That this represented an adaptive response, and not an adverse toxic effect, was indicated by enhanced survival of ribosome mutants after arsenic exposure. These results suggest that a major source of toxicity of trivalent arsenic derives from misfolding of newly synthesized proteins, and identifies ribosome reduction as a rapid, effective, and reversible proteotoxic stress response.

Histone deacetylase inhibitors target the leukemic microenvironment by enhancing a Nherf1-Protein Phosphatase 1{alpha}-TAZ signaling pathway in osteoblasts [Molecular Bases of Disease]

October 21st, 2015 by

Disrupting the protective signals provided by the bone marrow microenvironment will be critical for more effective combination drug therapies for acute myeloid leukemia (AML). Cells of the osteoblast lineage which reside in the endosteal niche have been implicated in promoting survival of AML cells. Here, we investigated how to prevent this protective interaction. We previously showed that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis of AML cells, unless the leukemic cells receive protective signals provided by differentiating osteoblasts. We now identify a novel signaling pathway in differentiating osteoblasts that can be manipulated in order to disrupt the osteoblast-mediated protection of AML cells. Treating differentiating osteoblasts with histone deacetylase inhibitors (HDACi) abrogated their ability to protect co-cultured AML cells from SDF-1-induced apoptosis. HDACi prominently up-regulated expression of the Nherf1 scaffold protein, which played a major role in preventing osteoblast-mediated protection of AML cells. Protein Phosphatase-1α (PP1α) was identified as a novel Nherf1 interacting protein that acts as the downstream mediator of this response by promoting nuclear localization of the TAZ transcriptional modulator. Moreover, independent activation of either PP1α or TAZ was sufficient to prevent osteoblast-mediated protection of AML cells even in the absence of HDACi. Together, these results indicate that HDACi target the AML microenvironment by enhancing activation of the Nherf1-PP1α-TAZ pathway in osteoblasts. Selective drug targeting of this osteoblast signaling pathway may improve treatments of AML by rendering leukemic cells in the bone marrow more susceptible to apoptosis.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Histone deacetylase inhibitors target the leukemic microenvironment by enhancing a Nherf1-Protein Phosphatase 1{alpha}-TAZ signaling pathway in osteoblasts [Molecular Bases of Disease]

Human telomerase reverse transcriptase (hTERT) transcription requires Sp1/Sp3 binding to the promoter and a permissive chromatin environment [DNA and Chromosomes]

October 20th, 2015 by Cheng, D., Zhao, Y., Wang, S., Jia, W., Kang, J., Zhu, J.

The transcription of human telomerase gene hTERT is regulated by transcription factors (TFs), including Sp1 family proteins, and its chromatin environment. To understand its regulation in a relevant chromatin context, we employed BAC reporters containing 160-kb of human genomic sequence containing the hTERT gene. Upon chromosomal integration, the BACs recapitulated endogenous hTERT expression, contrary to transient reporters. Sp1/Sp3 expression did not correlate with hTERT promoter activity and these TFs bound to the hTERT promoters in both telomerase-positive and -negative cells. Mutation of the proximal GC-box resulted in a dramatic decrease of hTERT promoter activity and mutations of all five GC-boxes eliminated its transcriptional activity. Neither mutations of GC-boxes nor knockdown of endogenous Sp1 impacted promoter binding by other TFs, including E-box binding proteins, and histone acetylation and trimethylation of histone H3K9 at the hTERT promoter in telomerase-positive and -negative cells. The result indicated that promoter binding by Sp1/Sp3 was essential, but not a limiting step, for hTERT transcription. hTERT transcription required a permissive chromatin environment. Importantly, our data also revealed different functions of GC-boxes and E-boxes in hTERT regulation: while GC-boxes were essential for promoter activity, factors bound to the E-boxes functioned to de-repress hTERT promoter.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Human telomerase reverse transcriptase (hTERT) transcription requires Sp1/Sp3 binding to the promoter and a permissive chromatin environment [DNA and Chromosomes]

A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence [Plant Biology]

October 20th, 2015 by Zhang, C., Muench, D. G.

PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins, and these show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for non-classical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was ten nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, as all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5[prime] end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of three of the ten predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred ten-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA processing. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.

C1q Deficiency Promotes Pulmonary Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury. [Cell Biology]

October 20th, 2015 by

The collectin proteins are innate immune molecules found in high concentrations on the epithelial and endothelial surfaces of the lung. While these proteins are known to have important anti-inflammatory actions in the airways of the lung little is known of their functional importance in the pulmonary circulation. We recently demonstrated that the circulating collectin protein adiponectin has potent anti-inflammatory effects on the lung endothelium, leading us to reason that other structurally-related proteins might have similar effects. To test this hypothesis, we investigated the anti-inflammatory actions of C1q in lung endothelial homeostasis and the pulmonary vascular response to LPS or HCl injury. We show that lung endothelium from C1q deficient (C1q-/-) mice expresses higher baseline levels of the vascular adhesion markers ICAM-1, VCAM-1 and E-selectin when compared to wild-type mice. Further, we demonstrate that these changes are associated with enhanced susceptibility of the lung to injury as evident by increased expression of adhesion markers, enhanced production of pro-inflammatory cytokines, and augmented neutrophil recruitment. Additionally, we found that C1q-/- mice also exhibited enhanced endothelial barrier dysfunction after injury as manifested by decreased expression of junctional adherens proteins and enhanced vascular leakage. Mechanistically, C1q appears to mediate its effects by inhibiting phosphorylation of p38 mitogen-activated protein kinase (MAPK) and blocking nuclear translocation of the P65 subunit of nuclear factor (NF)-kB. In summary, our findings indicate a previously unrecognized role for C1q in pulmonary vascular homeostasis and provide added support for the hypothesis that circulating collectin proteins have protective effects on the lung endothelium.

Normal Fertility Requires Expression of Carbonic Anhydrases II and IV in Sperm [Cell Biology]

October 20th, 2015 by

HCO3- is a key factor in the regulation of sperm motility. High concentrations of HCO3- in the female genital tract induce an increase in sperm beat frequency which speeds their progress through the female reproductive tract. Carbonic anhydrases (CA) which catalyze the reversible hydration of CO2 to HCO3-, represent potential candidates in the regulation of the HCO3- homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms - CAII and CAIV - are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find CAII is located in the principal piece whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3- homeostasis, resulting in substantially reduced sperm motility, swimming speed and HCO3--enhanced beat frequency. The CA activity remaining in sperm of CAII and CAIV-null mutants is 35% and 68% of that found for WT mice. Sperm of the double knockout mutant mice showed responses to stimulus by HCO3- or CO2 that were delayed in onset and reduced in magnitude. In contrast to sperm from double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3-. These results suggest that CAII and CAIV are required for optimal fertilization.

Defining the apoptotic trigger: the interaction of cytochrome c and cardiolipin [Membrane Biology]

October 20th, 2015 by O'Brien, E. S., Nucci, N. V., Fuglestad, B., Tommos, C., Wand, A. J.

The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (bb ~ 0.23 A) using NMR-based methods and is closely similar to the cryogenic crystal structure (bb ~ 1.2 A). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe36, Gly37, Thr58, Trp59, and Lys60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.

Twist1 is Essential for Tooth Morphogenesis and Odontoblast Differentiation [Molecular Bases of Disease]

October 20th, 2015 by

Twist1 is a basic helix-loop-helix (bHLH)- containing transcription factor that is expressed in the dental mesenchyme during the early stages of tooth development. To better delineate its roles in tooth development, we generated Twist1 conditional knockout embryos (Twist2Cre/+;Twist1fl/fl) by breeding Twist1 floxed mice (Twist1fl/fl) with Twist2-Cre knock-in mice (Twist2Cre/+). The Twist2Cre/+;Twist1fl/fl embryos formed smaller tooth germs and abnormal cusps during early tooth morphogenesis. Molecular and histological analyses showed that the developing molars of the Twist2Cre/+;Twist1fl/fl embryos had reduced cell proliferation and expression of fibroblast growth factors (Fgfs) 3, 4, 9 and 10, and FGF receptors (Fgfrs) 1 and 2 in the dental epithelium and mesenchyme. In addition, 3-week-old renal capsular transplants of the E18.5 Twist2Cre/+;Twist1fl/fl molars showed malformed crowns and cusps with defective crown dentin and enamel. Immunohistochemical analyses revealed that the implanted mutant molars had defects in odontoblast differentiation and delayed ameloblast differentiation. Furthermore, in vitro chromatin immuno-precipitation (ChIP) assays demonstrated that Twist1 was able to bind to a specific region of the Fgf10 promoter. In conclusion, our findings suggest that Twist1 plays crucial roles in regulating tooth development and that it may exert its functions through the FGF signaling pathway.

Humanized Affinity-Matured Monoclonal Antibody 8H9 Has Potent Anti-Tumor Activity and Binds to FG Loop of B7-H3 [Protein Structure and Folding]

October 20th, 2015 by

B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells, and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation and epitope mapping of 8H9 based on structure determination, modeling and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5 Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (ScFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescent cell sorting. Six mutations (three in the CDR and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nM KD) compared to parental hu8H9 scFv (144 nM KD). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to sub-nanomolar KD) had 2- to 9-fold enhancements in affinity compared to their parental forms, potent in vitro antibody dependent cell-mediated cytotoxicity (0.1-0.3 μg/mL EC50), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the precise molecular epitope of 8H9 was determined to be the FG loop of B7-H3, a region critical to its function in immunologic blockade, unique among anti-B7-H3 antibodies published to date.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Humanized Affinity-Matured Monoclonal Antibody 8H9 Has Potent Anti-Tumor Activity and Binds to FG Loop of B7-H3 [Protein Structure and Folding]

N-helix and cysteines inter-regulate human mitochondrial VDAC-2 function and biochemistry [Bioenergetics]

October 20th, 2015 by Maurya, S. R., Mahalakshmi, R.

Human voltage-dependent anion channel-2 (hVDAC-2) functions primarily as the crucial anti-apoptotic protein in the outer mitochondrial membrane, and additionally as a gated bidirectional metabolite transporter. The N-terminal helix (NTH), involved in voltage sensing, bears an additional 11-residue extension (NTE) only in hVDAC-2. In this study, we assign a unique role for the NTE as influencing the chaperone-independent refolding kinetics and overall thermodynamic stability of hVDAC-2. Our electrophysiology data shows that N-helix is crucial for channel activity while NTE sensitizes this isoform to voltage gating. Additionally, hVDAC-2 possesses the highest cysteine content, possibly for regulating reactive oxygen species content. We identify interdependent contributions of the N-helix and cysteines to channel function, and the measured stability in micellar environments with differing physico-chemical properties. The evolutionary demand for the NTE in the presence of cysteines clearly emerges from our biochemical and functional studies, providing insight into factors that functionally demarcate hVDAC-2 from the other VDACs.