For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.
Splice isoform and pharmacological studies reveal that sterol depletion relocalizes alpha-synuclein and enhances its toxicity.
Proc Natl Acad Sci U S A. 2014 Feb 25;111(8):3014-9.
Valastyan, J.S.,* Termine, D.J.*, and Lindquist, S.*
Synucleinopathies are neurodegenerative diseases associated with toxicity of the lipid-binding protein alpha-synuclein (alpha-syn). When expressed in yeast, alpha-syn associates with membranes at the endoplasmic reticulum and trafficks with vesicles out to the plasma membrane. At higher levels it elicits a number of phenotypes, including blocking vesicle trafficking. The expression of alpha-syn splice isoforms varies with disease, but how these isoforms affect protein function is unknown. We investigated two of the most abundant isoforms, resulting in deletion of exon four (alpha-synDelta4) or exon six (alpha-synDelta6). alpha-SynDelta4, missing part of the lipid-binding domain, had reduced toxicity and membrane binding. alpha-SynDelta6, missing part of the protein-protein interaction domain, had reduced toxicity but no reduction in membrane binding. To compare the mechanism by which the splice isoforms exert toxicity, equally toxic strains were probed with genetic modifiers of alpha-syn-induced toxicity. Most modifiers equally altered the toxicity induced by the splice isoforms and full-length alpha-syn (alpha-synFL). However, the splice isoform strains responded differently to a sterol-binding protein, leading us to examine the effect of sterols on alpha-syn-induced toxicity. Upon inhibition of sterol synthesis, alpha-synFL and alpha-synDelta6, but not alpha-synDelta4, showed decreased plasma membrane association, increased vesicular association, and increased cellular toxicity. Thus, higher membrane sterol concentrations favor plasma membrane binding of alpha-synFL and alpha-synDelta6 and may be protective of synucleinopathy progression. Given the common use of cholesterol-reducing statins and these potential effects on membrane binding proteins, further investigation of how sterol concentration and alpha-syn splice isoforms affect vesicular trafficking in synucleinopathies is warranted.
Distinct prion strains are defined by amyloid core structure and chaperone binding site dynamics.
Chem Biol. 2014 Feb 20;21(2):295-305.
Frederick, K.K.*, Debelouchina, G.T., Kayatekin, C.*, Dorminy, T., Jacavone, A.C., Griffin, R.G., and Lindquist, S.*
Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Using magic-angle spinning nuclear magnetic resonance spectroscopy, we provide a detailed look at the dynamic properties of these forms over a broad range of timescales. We establish that different prion strains have distinct amyloid structures, with many side chains in different chemical environments. Surprisingly, the prion strain with a larger fraction of rigid residues also has a larger fraction of highly mobile residues. Differences in mobility correlate with differences in interaction with the prion-partitioning factor Hsp104 in vivo, perhaps explaining strain-specific differences in inheritance.
N-acetyl-serotonin offers neuroprotection through inhibiting mitochondrial death pathways and autophagic activation in experimental models of Ischemic Injury.
J Neurosci. 2014 Feb 19;34(8):2967-78.
Zhou, H., Wang, J., Jiang, J., Stavrovskaya, I.G., Li, M., Li, W., Wu, Q., Zhang, X., Luo, C., Zhou, S., Sirianni, A.C,, Sarkar, S.*, Kristal, B.S., Friedlander, R.M., and Wang, X.
N-acetylserotonin (NAS) is an immediate precursor of melatonin, which we have reported is neuroprotective against ischemic injury. Here we test whether NAS is a potential neuroprotective agent in experimental models of ischemic injury. We demonstrate that NAS inhibits cell death induced by oxygen-glucose deprivation or H2O2 in primary cerebrocortical neurons and primary hippocampal neurons in vitro, and organotypic hippocampal slice cultures ex vivo and reduces hypoxia/ischemia injury in the middle cerebral artery occlusion mouse model of cerebral ischemia in vivo. We find that NAS is neuroprotective by inhibiting the mitochondrial cell death pathway and the autophagic cell death pathway. The neuroprotective effects of NAS may result from the influence of mitochondrial permeability transition pore opening, mitochondrial fragmentation, and inhibition of the subsequent release of apoptogenic factors cytochrome c, Smac, and apoptosis-inducing factor from mitochondria to cytoplasm, and activation of caspase-3, -9, as well as the suppression of the activation of autophagy under stress conditions by increasing LC3-II and Beclin-1 levels and decreasing p62 level. However, NAS, unlike melatonin, does not provide neuroprotection through the activation of melatonin receptor 1A. We demonstrate that NAS reaches the brain subsequent to intraperitoneal injection using liquid chromatography/mass spectrometry analysis. Given that it occurs naturally and has low toxicity, NAS, like melatonin, has potential as a novel therapy for ischemic injury.
Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells.
J Exp Med. 2014 Feb 10;211(2):365-79.
Avalos, A.M.*, Bilate, A.M.*, Witte, M.D.*, Tai, A.K., He, J., Frushicheva, M.P., Thill, P.D., Meyer-Wentrup, F.*, Theile, C.S.*, Chakraborty, A.K., Zhuang, X., and Ploegh, H.L.*
Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR. By altering length and valency of epitope-bearing synthetic peptides, we examined the properties of ligands required for optimal OB1 B cell activation. Monovalent engagement of the BCR with an epitope-bearing 17-mer synthetic peptide readily activated OB1 B cells. Dimers of the minimal peptide epitope oriented in an N to N configuration were more stimulatory than their C to C counterparts. Although shorter length correlated with less activation, a monomeric 8-mer peptide epitope behaved as a weak agonist that blocked responses to cell-bound peptide antigen, a blockade which could not be reversed by CD40 ligation. The 8-mer not only delivered a suboptimal signal, which blocked subsequent responses to OVA, anti-IgG, and anti-kappa, but also competed for binding with OVA. Our results show that fine-tuning of BCR-ligand recognition can lead to B cell nonresponsiveness, activation, or inhibition.
In vivo discovery of immunotherapy targets in the tumour microenvironment.
Nature. 2014 Feb 6;506(7486):52-7.
Zhou, P., Shaffer, D.R., Alvarez Arias, D.A., Nakazaki, Y., Pos, W., Torres, A.J., Cremasco, V., Dougan, S.K.*, Cowley, G.S., Elpek, K., Brogdon, J., Lamb, J., Turley, S.J., Ploegh, H.L.*, Root, D.E., Love, J.C., Dranoff, G., Hacohen, N., Cantor, H., and Wucherpfennig, K.W.
Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.
The master regulator of the cellular stress response (HSF1) is critical for orthopoxvirus infection.
PLoS Pathog. 2014 Feb 6;10(2):e1003904.
Filone, C.M., Caballero, I.S., Dower, K., Mendillo, M.L.*, Cowley, G.S., Santagata, S.*, Rozelle, D.K., Yen, J., Rubins, K.H.*, Hacohen, N., Root, D.E., Hensley, L.E., and Connor, J.
The genus Orthopoxviridae contains a diverse group of human pathogens including monkeypox, smallpox and vaccinia. These viruses are presumed to be less dependent on host functions than other DNA viruses because they have large genomes and replicate in the cytoplasm, but a detailed understanding of the host factors required by orthopoxviruses is lacking. To address this topic, we performed an unbiased, genome-wide pooled RNAi screen targeting over 17,000 human genes to identify the host factors that support orthopoxvirus infection. We used secondary and tertiary assays to validate our screen results. One of the strongest hits was heat shock factor 1 (HSF1), the ancient master regulator of the cytoprotective heat-shock response. In investigating the behavior of HSF1 during vaccinia infection, we found that HSF1 was phosphorylated, translocated to the nucleus, and increased transcription of HSF1 target genes. Activation of HSF1 was supportive for virus replication, as RNAi knockdown and HSF1 small molecule inhibition prevented orthopoxvirus infection. Consistent with its role as a transcriptional activator, inhibition of several HSF1 targets also blocked vaccinia virus replication. These data show that orthopoxviruses co-opt host transcriptional responses for their own benefit, thereby effectively extending their functional genome to include genes residing within the host DNA. The dependence on HSF1 and its chaperone network offers multiple opportunities for antiviral drug development.
Toxoplasma gondii inhibits gamma interferon (IFN-gamma)- and IFN-beta-induced host cell STAT1 transcriptional activity by increasing the association of STAT1 with DNA.
Infect Immun. 2014 Feb;82(2):706-19.
Rosowski, E.E., Nguyen, Q.P., Camejo, A., Spooner, E.*, and Saeij, J.P.
The gamma interferon (IFN-gamma) response, mediated by the STAT1 transcription factor, is crucial for host defense against the intracellular pathogen Toxoplasma gondii, but prior infection with Toxoplasma can inhibit this response. Recently, it was reported that the Toxoplasma type II NTE strain prevents the recruitment of chromatin remodeling complexes containing Brahma-related gene 1 (BRG-1) to promoters of IFN-gamma-induced secondary response genes such as Ciita and major histocompatibility complex class II genes in murine macrophages, thereby inhibiting their expression. We report here that a type I strain of Toxoplasma inhibits the expression of primary IFN-gamma response genes such as IRF1 through a distinct mechanism not dependent on the activity of histone deacetylases. Instead, infection with a type I, II, or III strain of Toxoplasma inhibits the dissociation of STAT1 from DNA, preventing its recycling and further rounds of STAT1-mediated transcriptional activation. This leads to increased IFN-gamma-induced binding of STAT1 at the IRF1 promoter in host cells and increased global IFN-gamma-induced association of STAT1 with chromatin. Toxoplasma type I infection also inhibits IFN-beta-induced interferon-stimulated gene factor 3-mediated gene expression, and this inhibition is also linked to increased association of STAT1 with chromatin. The secretion of proteins into the host cell by a type I strain of Toxoplasma without complete parasite invasion is not sufficient to block STAT1-mediated expression, suggesting that the effector protein responsible for this inhibition is not derived from the rhoptries.
Poly(A)-tail profiling reveals an embryonic switch in translational control.
Nature. 2014 Jan 29.
Subtelny, A.O.*, Eichhorn, S.W.*, Chen, G.R.*, Sive, H.*, and Bartel, D.P.*
Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.
Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis.
J Cell Sci. 2014 Jan 15;127(Pt 2):445-54.
Tafesse, F.G.*, Vacaru, A.M., Bosma, E.F., Hermansson, M., Jain, A., Hilderink, A., Somerharju, P., and Holthuis, J.C.M.
Cells synthesize ceramides in the endoplasmic reticulum (ER) as precursors for sphingolipids to form an impermeable plasma membrane. As ceramides are engaged in apoptotic pathways, cells would need to monitor their levels closely to avoid killing themselves during sphingolipid biosynthesis. How this is accomplished remains to be established. Here we identify SMSr (SAMD8), an ER-resident ceramide phosphoethanolamine (CPE) synthase, as a suppressor of ceramide-mediated cell death. Disruption of SMSr catalytic activity causes a rise in ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Blocking de novo ceramide synthesis, stimulating ceramide export from the ER or targeting a bacterial ceramidase to mitochondria rescues SMSr-deficient cells from apoptosis. We also show that SMSr-catalyzed CPE production, although essential, is not sufficient to suppress ceramide-induced cell death and that SMSr-mediated ceramide homeostasis requires the N-terminal sterile alpha-motif, or SAM domain, of the enzyme. These results define ER ceramides as bona fide transducers of mitochondrial apoptosis and indicate a primary role of SMSr in monitoring ER ceramide levels to prevent inappropriate cell death during sphingolipid biosynthesis.
Genome-wide localization of small molecules.
Nat Biotechnol. 2014 Jan;32(1):92-6.
Anders, L.*, Guenther, M.G.*, Qi, J.*, Fan, Z.P.*, Marineau, J.J., Rahl, P.B.*, Loven, J.*, Sigova, A.A.*, Smith, W.B., Lee, T.I.*, Bradner, J.E., and Young, R.A.*
A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.
*Author affiliated with Whitehead Institute for Biomedical Research