Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

National Institutes of Health

Human La protein has been implicated in facilitating internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV). Earlier, we demonstrated that the RNA recognition motif (RRM) encompassing residues 112 to 184 of La protein [La (112-184)] interacts with the HCV IRES near the initiator AUG codon. A synthetic peptide, LaR2C (24-mer), derived from La RRM (112-184), retains RNA binding ability, competes with La protein binding to the HCV IRES, and inhibits translation. The peptide interferes with the assembly of 48S complexes, resulting in the accumulation of preinitiation complexes that are incompetent for the 60S ribosomal subunit joining. Here, nuclear magnetic resonance spectroscopy of the HCV IRES-bound peptide complex revealed putative contact points, mutations that showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM (112-184) structure. A 7-mer peptide comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred nuclear Overhauser effect experiments suggests that it is a beta turn. This conformation is significantly different from that observed in the free RRM (112-184) NMR structure, suggesting paths toward a better-stabilized mimetic peptide. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition of HCV IRES function. More importantly, the peptide significantly inhibited replication of the HCV monocistronic replicon. Elucidation of the structural determinant of the peptide provides a basis for developing small peptidomimetic structures as potent anti-HCV therapeutics.

Structural determinant of human La protein critical for internal initiation of translation of hepatitis C virus RNA.

&#xB54C;&#xBB38;&#xC5D0; &#xC81C;&#xD55C; &#xB41C; &#xC0AC;&#xC6A9; &#xAC00;&#xB2A5;&#xD55C; &#xCE58;&#xB8CC; &#xC635;&#xC158;, C &#xD615; &#xAC04;&#xC5FC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xB9AC;&#xB4DC; &#xD654;&#xD569;&#xBB3C; &#xAC1C;&#xBC1C; &#xC2DC;&#xAE09;&#xC740;. &#xC778;&#xAC04;&#xC758; &#xB77C; &#xB2E8;&#xBC31;&#xC9C8; C &#xD615; &#xAC04;&#xC5FC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC640; C &#xD615; &#xAC04;&#xC5FC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; RNA&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xD1B5;&#xD574; &#xBC88;&#xC5ED;&#xC744; &#xC790;&#xADF9;. &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; RNA&#xC640; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD558;&#xB294; &#xC778;&#xAC04;&#xC758; &#xB77C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &beta;-&#xD134;&#xC744; &#xD749;&#xB0B4; &#xB0B8; &#xC8FC;&#xAE30;&#xC801; &#xD3A9; &#xD2F0; &#xB4DC;&#xB97C; &#xD569;&#xC131; &#xD588;&#xB2E4;. C &#xD615; &#xAC04;&#xC5FC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; RNA &#xBC88;&#xC5ED;&#xC744; &#xD574;&#xB2F9; &#xC120;&#xD615; &#xD3A9; &#xD2F0; &#xB4DC; &#xBCF4;&#xB2E4; &#xD6E8;&#xC52C; &#xB354; &#xC774;&#xC0C1; &#xD6C4; &#xCC98;&#xB9AC; &#xC2DC;&#xAC04;&#xC744; &#xC5B5;&#xC81C; &#xD55C;&#xB2E4;. Replicon &#xC2E4;&#xC2DC;&#xAC04; RT-PCR&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; RNA &#xC218;&#xC900;&#xC744; &#xCE21;&#xC815; &#xC8FC;&#xAE30;&#xC801; &#xD3A9; &#xD2F0; &#xB4DC;&#xB3C4; &#xBCF5;&#xC81C;&#xB97C; &#xAE08; &#xD588;&#xB2E4;. &#xC8FC;&#xAE30;&#xC801; &#xD3A9; &#xD2F0; &#xB4DC; &#xD615; &#xAC04;&#xC5FC; c.&#xC5D0; &#xB300; &#xD55C; &#xBCF5;&#xD569; &#xC720;&#xB9DD;&#xD55C; &#xB9AC;&#xB4DC;&#xB85C; &#xB098;&#xC628;&#xB2E4;

&#xC778;&#xAC04;&#xC758; &#xB77C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; RNA &#xC778;&#xC2DD; &#xBAA8;&#xD2F0;&#xBE0C;&#xC758; &#xC8FC;&#xAE30;&#xC801; &#xD3A9;&#xD0C0;&#xC774;&#xB4DC; &#xBAA8;&#xBC29;&#xC740; C &#xD615; &#xAC04;&#xC5FC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xAC15;&#xB825;&#xD55C; &#xC5B5;&#xC81C;&#xC81C; &#xC774;&#xB2E4;.

Aufgrund der begrenzt verf&uuml;gbaren therapeutischen Optionen ist die Entwicklung neuer Bleiverbindungen gegen Hepatitis C-Virus dringend notwendig. Menschliche La Protein stimuliert Hepatitis C-Virus-&Uuml;bersetzung durch Interaktion mit dem Hepatitis-C-Virus-RNA. Ein zyklisches Peptid imitiert das &beta;-Turn des menschlichen La Proteins, die Interaktion mit der viralen RNA wurde synthetisiert. Es hemmt die Hepatitis C virale RNA &Uuml;bersetzung wesentlich besser als die entsprechenden linearen Peptid zu mehr Nachbehandlung Zeiten. Das zyklische Peptid hemmte auch Replikation Replikon RNA-Levels, die mit Real-Time RT-PCR gemessen. Das zyklische Peptid entpuppt sich als eine vielversprechende Blei zusammengesetzte gegen Hepatitis c.

Eine zyklische Peptid-Nach&auml;ffer ein RNA-Anerkennung-Motiv des menschlichen Proteins in La ist ein potenter Inhibitor der Hepatitis-C-Virus.

A causa di limitate opzioni terapeutiche disponibili, sviluppare nuovi composti di piombo contro il virus dell&#39;epatite C &egrave; un&#39;urgente necessit&agrave;. La proteina umana stimola traduzione virus dell&#39;epatite C attraverso l&#39;interazione con il RNA virale dell&#39;epatite C. &Egrave; stato sintetizzato un peptide ciclico che mimano la &beta;-girata della umana La proteina che interagisce con il RNA virale. Inibisce l&#39;epatite C virale RNA traduzione significativamente migliore rispetto al peptide lineare corrispondente a pi&ugrave; tempi di post-trattamento. Il peptide ciclico inibisce anche la replica come misurato da replicone livelli di RNA mediante RT-PCR in tempo reale. Il peptide ciclico emerge come un promettente lead compound contro epatite c.

Una mimica del peptide ciclico di un motivo di riconoscimento RNA umano La proteina &egrave; un potente inibitore del virus dell&#39;epatite C.

&#x9650;&#x3089;&#x308C;&#x305F;&#x5229;&#x7528;&#x53EF;&#x80FD;&#x306A;&#x6CBB;&#x7642;&#x30AA;&#x30D7;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x305F;&#x3081;&#x3001;&#x65B0;&#x3057;&#x3044;&#x30EA;&#x30FC;&#x30C9;&#x5316;&#x5408;&#x7269; C &#x578B;&#x809D;&#x708E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x958B;&#x767A;&#x304C;&#x7DCA;&#x6025;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x4EBA;&#x9593;&#x306E;&#x30E9;&#x86CB;&#x767D;&#x8CEA; C &#x578B;&#x809D;&#x708E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x7FFB;&#x8A33;&#x306B;&#x3088;&#x308B; C &#x578B;&#x809D;&#x708E;&#x30A6;&#x30A4;&#x30EB;&#x30B9; RNA &#x3068;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x523A;&#x6FC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E; RNA &#x3068;&#x5BFE;&#x8A71;&#x3059;&#x308B;&#x4EBA;&#x9593;&#x306E;&#x30E9;&#x86CB;&#x767D;&#x8CEA;&#x306E; &beta; &#x30BF;&#x30FC;&#x30F3;&#x3092;&#x6A21;&#x5023;&#x74B0;&#x72B6;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x3092;&#x5408;&#x6210;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;C &#x578B;&#x809D;&#x708E;&#x30A6;&#x30A4;&#x30EB;&#x30B9; RNA &#x7FFB;&#x8A33;&#x5BFE;&#x5FDC;&#x3059;&#x308B;&#x7DDA;&#x5F62;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x3088;&#x308A;&#x3082;&#x5927;&#x5E45;&#x306B;&#x5411;&#x4E0A;&#x5F8C;&#x51E6;&#x7406;&#x6642;&#x3082;&#x306F;&#x3084;&#x3092;&#x963B;&#x5BB3;&#x3057;&#x307E;&#x3059;&#x3002;&#x74B0;&#x72B6;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x306F;&#x3001;&#x307E;&#x305F;&#x30EC;&#x30D7;&#x30EA;&#x30B3;&#x30F3;&#x306B;&#x3088;&#x308B;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0; RT-PCR &#x6CD5;&#x3092;&#x7528;&#x3044;&#x305F; RNA &#x30EC;&#x30D9;&#x30EB;&#x6E2C;&#x5B9A;&#x3068;&#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x6291;&#x5236;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x74B0;&#x72B6;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x304C;&#x6709;&#x671B;&#x306A;&#x6F5C;&#x5728;&#x9867;&#x5BA2;&#x3068;&#x3057;&#x3066;&#x306E; c &#x578B;&#x809D;&#x708E;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x8907;&#x5408;&#x51FA;&#x73FE;&#x3057;&#x307E;&#x3059;&#x3002;

&#x74B0;&#x72B6;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x6A21;&#x5023;&#x4EBA;&#x9593;&#x30E9;&#x86CB;&#x767D;&#x8CEA;&#x306E; RNA &#x8A8D;&#x8B58;&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306E; C &#x578B;&#x809D;&#x708E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x5F37;&#x529B;&#x306A;&#x963B;&#x5BB3;&#x5264;&#x3067;&#x3059;&#x3002;

&#x7531;&#x4E8E;&#x53EF;&#x7528;&#x6CBB;&#x7597;&#x9009;&#x62E9;&#x6709;&#x9650;&#xFF0C;&#x53D1;&#x5C55;&#x65B0;&#x7684;&#x94C5;&#x5316;&#x5408;&#x7269;&#x6297;&#x4E19;&#x578B;&#x809D;&#x708E;&#x75C5;&#x6BD2;&#x662F;&#x8FEB;&#x5207;&#x7684;&#x9700;&#x8981;&#x3002;&#x4EBA; La &#x86CB;&#x767D;&#x523A;&#x6FC0;&#x4E19;&#x578B;&#x809D;&#x708E;&#x75C5;&#x6BD2;&#x901A;&#x8FC7;&#x4E0E;&#x4E19;&#x578B;&#x809D;&#x708E;&#x75C5;&#x6BD2; RNA &#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684;&#x7FFB;&#x8BD1;&#x3002;&#x6A21;&#x4EFF;&#x7684;&#x75C5;&#x6BD2; RNA &#x8FDB;&#x884C;&#x4EA4;&#x4E92;&#x4EBA; La &#x86CB;&#x767D; &beta;-&#x4EA4;&#x66FF;&#x5FAA;&#x73AF;&#x80BD;&#x88AB;&#x5408;&#x6210;&#x3002;&#x5B83;&#x6291;&#x5236;&#x4E19;&#x578B;&#x809D;&#x708E;&#x75C5;&#x6BD2; RNA &#x7FFB;&#x8BD1;&#x660E;&#x663E;&#x4F18;&#x4E8E;&#x76F8;&#x5E94;&#x7684;&#x7EBF;&#x6027;&#x591A;&#x80BD;&#x5728;&#x540E;&#x5904;&#x7406;&#x65F6;&#x95F4;&#x957F;&#x3002;&#x5FAA;&#x73AF;&#x7684;&#x591A;&#x80BD;&#x4E5F;&#x6291;&#x5236;&#x590D;&#x5236;&#x590D;&#x5236;&#x5B50;&#x4F7F;&#x7528;&#x5B9E;&#x65F6;&#x9006;&#x8F6C;&#x5F55;-&#x805A;&#x5408;&#x9176;&#x94FE;&#x53CD;&#x5E94;&#x7684; RNA &#x6C34;&#x5E73;&#x6765;&#x8861;&#x91CF;&#x3002;&#x5FAA;&#x73AF;&#x7684;&#x80BD;&#x6210;&#x4E3A;&#x6709;&#x524D;&#x9014;&#x7684;&#x94C5;&#x590D;&#x65B9;&#x6297;&#x809D;&#x708E; C.

&#x4EBA; La &#x86CB;&#x767D;&#x7684; RNA &#x8BC6;&#x522B;&#x57FA;&#x5E8F;&#x5FAA;&#x73AF;&#x80BD;&#x6A21;&#x4EFF;&#x662F;&#x4E00;&#x79CD;&#x5F3A;&#x6548;&#x6291;&#x5236;&#x5242; C &#x578B;&#x809D;&#x708E;&#x75C5;&#x6BD2;&#x3002;

Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the Î²-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

A cyclic peptide mimic of an RNA recognition motif of human La protein is a potent inhibitor of hepatitis C virus.

En raison des options th&eacute;rapeutiques disponibles limit&eacute;es, le d&eacute;veloppement de nouveaux compos&eacute;s de plomb contre le virus de l&#39;h&eacute;patite C est urgent. Stimule la prot&eacute;ine humaine de La traduction de virus de l&#39;h&eacute;patite C gr&acirc;ce &agrave; l&#39;interaction avec l&#39;ARN viral de l&#39;h&eacute;patite C. Un peptide cyclique imitant le coude-&beta; de la prot&eacute;ine humaine de La qui interagissent avec l&#39;ARN viral a &eacute;t&eacute; synth&eacute;tis&eacute;. Il inhibe l&#39;h&eacute;patite C virale RNA traduction nettement mieux que le peptide lin&eacute;aire correspondant &agrave; des moments plus apr&egrave;s le traitement. Le peptide cyclique inhibe &eacute;galement la r&eacute;plication telle que mesur&eacute;e par r&eacute;plicon niveaux d&#39;ARN &agrave; l&#39;aide de RT-PCR en temps r&eacute;el. Le peptide cyclique appara&icirc;t comme une piste prometteuse compos&eacute; contre l&#39;h&eacute;patite c.

Un imitateur de peptide cyclique d&#39;un motif de reconnaissance de RNA des humains de La prot&eacute;ine est un puissant inhibiteur du virus de l&#39;h&eacute;patite C.

Debido a la limitada opciones terap&eacute;uticas disponibles, desarrollo de nuevos compuestos de plomo contra el virus de la hepatitis C es una necesidad urgente. Estimula la prote&iacute;na humana de La traducci&oacute;n de virus de hepatitis C a trav&eacute;s de la interacci&oacute;n con el RNA viral de la hepatitis C. Fue sintetizado un p&eacute;ptido c&iacute;clico m&iacute;mico la &beta;-vuelta de la prote&iacute;na humana de La que interact&uacute;a con el RNA viral. Inhibe la hepatitis C viral RNA traducci&oacute;n significativamente mejor que el p&eacute;ptido lineal correspondiente momentos ya despu&eacute;s del tratamiento. El p&eacute;ptido c&iacute;clico tambi&eacute;n inhibe la replicaci&oacute;n medida por replic&oacute;n niveles de ARN usando RT-PCR en tiempo real. El p&eacute;ptido c&iacute;clico emerge como un prometedor plomo compuesto contra la hepatitis C.

Un imitador de p&eacute;ptido c&iacute;clico de un motivo de reconocimiento del RNA de la prote&iacute;na de La humana es un potente inhibidor del virus de la hepatitis C.

Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a β-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting β-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La β-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein.

Specific sequence of a Beta turn in human la protein may contribute to species specificity of hepatitis C virus.

Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development.

Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and <0.01% cross-talk. Combining these technical advances affords the ability to move beyond obtaining superresolved structures to defining spatial relationships among constituent molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM.

Asit Manna

Laboratory of Cellular & Molecular Biology,
National Cancer Institute,
Laboratory of Cellular & Molecular Biology, National Cancer Institute

National Institutes of Health

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Asit Manna

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Cellular & Molecular Biology,National Cancer Institute,Laboratory of Cellular & Molecular Biology, National Cancer Institute

National Institutes of Health

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Laboratory of Cellular & Molecular Biology,
National Cancer Institute,
Laboratory of Cellular & Molecular Biology, National Cancer Institute