MicroRNA-7 Compromises p53-dependent Apoptosis by Controlling the Expression of the Chromatin Remodeling Factor SMARCD1 [Signal Transduction]

November 5th, 2015 by Hong, C.-F., Lin, S.-Y., Chou, Y.-T., Wu, C.-W.

We previously demonstrated that EGFR upregulated miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. In this study, we identified SMARCD1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. We used luciferase reporters carrying wild type or mutated 3'UTR of SMARCD1, and found that miR-7 blocked SMARCD1 expression by binding to two seed regions in the 3'UTR of SMARCD1 and downregulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 downregulated p53-dependent apoptosis-related gene BAX (BCL2-associated X protein) and p21 expression by interfering the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. We found that while SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was downregulated in non-small cell lung cancer (NSCLC) patients and lung adenocarcinoma cell lines, and SMARCD1 and miR-7 expression levels were negatively correlated in clinical samples. Our investigation into the involvement of the EGFR-regulated miRNA pathway in the SWI/SNF chromatin remodeling complex suggest that EGFR-mediated miR-7 suppressed the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells.
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Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium Independent Membrane Binding [Molecular Biophysics]

November 5th, 2015 by

Perforin is an essential component in the cytotoxic lymphocyte mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process, and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. While many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.
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The voltage-dependent anion channel 1 mediates amyloid beta toxicity and represents a potential target for Alzheimer’s disease therapy [Cell Biology]

November 5th, 2015 by

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is over-expressed in post-mortem brains of Alzheimer's disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid beta (Aβ). In this study, we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance and microscale thermophoresis. Pre-incubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance about two-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol, as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization and cytochrome release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial- and plasma membrane-VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.
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Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex [Cell Biology]

November 5th, 2015 by Mattera, R., Guardia, C. M., Sidhu, S., Bonifacino, J. S.

The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, however, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation. [Protein Synthesis and Degradation]

November 5th, 2015 by

Runx2, a master regulator of osteoblast differentiation is tightly regulated both at transcriptional and post-translational levels. Post translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, over-expression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function, however; both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized animals (induction of surgical menopause) that show reduced Runx2 and enhanced Fbw7α while this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before.
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Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-Chain Fatty Acyl-CoAs and a Novel CoA Induced Fit Mechanism [Enzymology]

November 4th, 2015 by

PaaI thioesterases are members of the TE13 thioesterase family which catalyse the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8 Å resolution, to reveal two double hotdog domains arranged in a back-to-back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle X-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn37, Asp52, and Thr68 are important for catalysis, and size exclusion chromatography analysis and X-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N-terminus, and a loop region involving Tyr38Tyr39. This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.
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Validation and Characterisation of a Novel Peptide that Binds Monomeric and Aggregated Beta-amyloid and Inhibits the Formation of Neurotoxic Oligomers [Molecular Bases of Disease]

November 4th, 2015 by

Although the formation of beta-amyloid (Aβ) deposits in the brain is a hallmark of Alzheimer's Disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aβ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble (Aβ) oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15 a.a peptide (15mer), isolated via phage display screening, targeted Aβ and attenuated its neurotoxicity (1). The aim of the current study was to generate and biochemically characterise analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15 a.a. peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t=300min c.f. t=30min for the parent peptide). This candidate reduced the formation of soluble Aβ42 oligomers with the concurrent generation of non-toxic insoluble aggregates measuring up to 25-30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aβ42, as shown by coimmunoprecipitation and Surface Plasmon Resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aβ42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aβ42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies.
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Bag2 Mediated Regulation of Pink1 is Critical for Mitochondrial Translocation of Parkin and Neuronal Survival [Molecular Bases of Disease]

November 4th, 2015 by

Emerging evidence has demonstrated a growing genetic component in Parkinson′s disease (PD). For instance, loss of function mutations in Pink1 or Parkin can cause autosomal recessive PD. Recently, Pink1 and Parkin have been implicated in the same signaling pathway to regulate mitochondrial clearance through recruitment of Parkin by stabilization of Pink1 on the outer membrane of depolarized mitochondria. The precise mechanisms that govern this process remain enigmatic. In this study, we identify Bcl2-associated athanogene 2 (Bag2) as a factor that promotes mitophagy. Bag2 inhibits Pink1 degradation by blocking the ubiquitinylation pathway. Stabilization of Pink1 by Bag2 triggers Parkin-mediated mitophagy and protects neurons against MPP+-induced oxidative stress in an in vitro cell model of PD. Collectively, our findings support the notion that Bag2 is an upstream regulator of the Pink1/Parkin signaling pathway.

Cleavage of signal regulatory protein alpha (SIRP{alpha}) enhances inflammatory signaling. [Immunology]

November 3rd, 2015 by Londino, J. D., Gulick, D., Isenberg, J. S., Mallampalli, R. K.

Signal regulatory protein alpha (SIRP α) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein CD47 results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs), resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and in human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane proximal domain necessary for ADAM10 mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.

The Metastasis Suppressor, N-myc Downstream Regulated Gene-1 (NDRG1), Down-Regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways [Cell Biology]

November 3rd, 2015 by

N-myc downstream regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways including, the transforming growth factor-beta; (TGF-beta) pathway, protein kinase B (AKT)/phosphatidylinositol-3-kinase (PI3K) pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) were examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2 and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream mitogen-activated protein kinase kinase (MAPKK) in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2 and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for Dp44mT, but was independent of this metastasis suppressor for DpC. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.
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