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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Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase [Microbiology]

November 3rd, 2015 by Maksimov, M. O., Koos, J. D., Zong, C., Lisko, B., Link, A. J.

Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. In order to carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduce cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.

A heme-responsive regulator controls synthesis of staphyloferrin B in Staphylococcus aureus [Gene Regulation]

November 3rd, 2015 by

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A (SA) and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it itself does not synthesize. The Ferric Uptake Regulator, or Fur, protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron-regulation via Fur, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onwards. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme, and that heme-bound SbnI does not bind DNA. Last, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.

HIV-1-Tat Inhibits SC35-Mediated Tau Exon 10 Inclusion through Upregulation of DYRK1A Kinase [Signal Transduction]

November 3rd, 2015 by

The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of Tau exon 10 results in tauopathies characterized by alterations in the proportion of Tau isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the Tau molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes Tau 3R upregulation though increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with Tau RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of Tau exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.

Exosome adherence and internalization by hepatic stellate cells triggers sphingosine 1-phosphate dependent migration [Cell Biology]

November 3rd, 2015 by

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC) derived exosomes could regulate the phenotype of hepatic stellate cells (HSC). Initial microarray studies showed fibroblast growth factor-2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1 overexpressing EC line increased HSC migration by 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSC with exosomes was also associated with an 8.3-fold increased phosphorylation of AKT and 2.5-fold increased migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot and transmission electron microscopy. Blockade of FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibitions of endocytosis with transfection of dynamin siRNA, dominant negative dynamin GTPase construct Dyn2K44A, or by the pharmacological inhibitor Dynasore, significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis and SK1 mRNA levels were upregulated in human liver cirrhosis patient samples by 2.5-fold. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Thus, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin dependent exosome adherence and dynamin dependent exosome internalization. These findings advance our understanding of EC/HSC crosstalk and identify exosomes as a potential target to attenuate pathobiology signals.

Time-resolved studies of IsdG identify molecular signposts along the non-canonical heme oxygenase pathway [Microbiology]

November 3rd, 2015 by

IsdGs are heme monooxygenases that break open the tetrapyrrole, releasing the iron and thereby allowing bacteria expressing this protein to use heme as a nutritional iron source. Little is currently known about the mechanism by which IsdGs degrade heme, though the products differ from those generated by canonical heme oxygenases. A synthesis of time resolved techniques including in proteo mass spectrometry and conventional and stopped flow UV/vis was used in conjunction with analytical methods to define the reaction steps mediated by IsdG from Staphylococcus aureus and their time scales. An apparent meso -(hydr)oxyheme (forming with k = 0.6 min-1, pH 7.4, 10 mM ascorbate, 10 μM IsdG-heme, 22 °C) was identified as a likely common intermediate with the canonical heme oxygenases (HOs). Unlike HOs, this intermediate does not form with added H2O2, nor does it convert to verdoheme and CO. Rather, the next observable intermediates (k = 0.16 min-1) were a set of formyl-oxo-bilin isomers, similar to the mycobilin products of the IsdG homolog from Mycobacterium tuberculosis (MhuD). These converted in separate fast and slow phases to β-/δ-staphylobilin isomers and formaldehyde (CH2O). Controlled release of this unusual C1 product may support IsdG′s dual role as both an oxygenase and a sensor of heme availability in S. aureus.

Immunometabolism; cellular metabolism turns immune regulator [Metabolism]

November 3rd, 2015 by Loftus, R. M., Finlay, D. K.

Immune cells are highly dynamic in terms of their growth, proliferation and effector functions as they respond to immunological challenges. Different immune cells can adopt distinct metabolic configurations that allow the cell to balance its requirements for energy, molecular biosynthesis and longevity. However, in addition to facilitating immune cell responses, it is now becoming clear that cellular metabolism has direct roles in regulating immune cell function. This review article describes the distinct metabolic signatures of key immune cells, explains how these metabolic setups facilitate immune function and discusses the emerging evidence that intracellular metabolism has an integral role in controlling immune responses.