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The coreceptor CD8αβ can greatly promote activation of T cells by strengthening T-cell receptor (TCR) binding to cognate peptide-MHC complexes (pMHC) on antigen presenting cells and by bringing p56Lck to TCR/CD3. Here, we demonstrate that CD8 can also bind to pMHC on the T cell (in cis) and that this inhibits their activation. Using molecular modeling, fluorescence resonance energy transfer experiments on living cells, biochemical and mutational analysis, we show that CD8 binding to pMHC in cis involves a different docking mode and is regulated by posttranslational modifications including a membrane-distal interchain disulfide bond and negatively charged O-linked glycans near positively charged sequences on the CD8β stalk. These modifications distort the stalk, thus favoring CD8 binding to pMHC in cis. Differential binding of CD8 to pMHC in cis or trans is a means to regulate CD8+ T-cell responses and provides new translational opportunities.

Telomeres are transcribed into telomeric repeat-containing RNA (TERRA), large, heterogeneous, noncoding transcripts which form part of the telomeric heterochromatin. Despite a large number of functions that have been ascribed to TERRA, little is known about its biogenesis. Here, we present the first comprehensive analysis of the molecular structure of TERRA. We identify biochemically distinct TERRA complexes, and we describe TERRA regulation during the cell cycle. Moreover, we demonstrate that TERRA 5' ends contain 7-methylguanosine cap structures and that the poly(A) tail, present on a fraction of TERRA transcripts, contributes to their stability. Poly(A)(-) TERRA, but not poly(A)(+) TERRA, is associated with chromatin, possibly reflecting distinct biological roles of TERRA ribonucleoprotein complexes. In support of this idea, poly(A)(-) and poly(A)(+) TERRA molecules end with distinct sequence registers. We also determine that the bulk of 3'-terminal UUAGGG repeats have an average length of 200 bases, indicating that the length heterogeneity of TERRA likely stems from its subtelomeric regions. Finally, we find that TERRA is regulated during the cell cycle, being lowest in late S phase and peaking in early G(1). Our analyses offer the basis for investigating multiple regulatory pathways that affect TERRA synthesis, processing, turnover, and function.

Telomerase is required for telomere maintenance and is responsible for the immortal phenotype of cancer cells. How telomerase is assembled and reaches telomeres in the context of nuclear architecture is not understood. Recently, the telomerase RNA subunit (hTR) was shown to accumulate in Cajal bodies (CBs), subnuclear structures implicated in ribonucleoprotein maturation. However, the functional relevance of this localization for telomerase was unknown. hTR localization to CBs requires a short sequence motif called the CAB box. Here, we reconstitute telomerase in human cells and determine the effects of CAB box mutations on telomere biology. We demonstrate that mutant hTR, which fails to accumulate in CBs, is fully capable of forming catalytically active telomerase in vivo but is strongly impaired in telomere extension. The functional deficiency is accompanied by a decreased association of telomerase with telomeres. Collectively, these data identify subnuclear localization as an important regulatory mechanism for telomere length homeostasis in human cells.

By using the Telospot assay, 27 different extracts of cyanobacteria were evaluated for telomerase inhibition. All extracts showed varying, but significant activity. We selected Microcystis aeruguinosa PCC 7806 to identify the active compound and a bioassay guided fractionation led us to isolate mixtures of sulfoquinovosyl diacylglycerols (SQDGs), which were identified by 2D NMR and MS/MS experiments. Pure SQDG derivatives were then synthesized. The IC(50) values of pure synthetic sulfoquinovosyl dipalmitoylglycerol and the monopalmitoylated derivative against telomerase were determined to be 17 and 40 μM, respectively. A structure-activity relationship study allowed the identification of compounds with modified lipophilic acyl groups that display improved activity.

Telomerase is responsible for the immortal phenotype of cancer cells and telomerase inhibition may specifically target cancer cell proliferation. Ligands able to selectively bind to G-quadruplex telomeric DNA have been considered as telomerase inhibitors but their mechanisms of action have often been deduced from a non-quantitative telomerase activity assay (TRAP assay) that involves a PCR step and that does not provide insight on the mechanism of inhibition. Furthermore, quadruplex ligands have also been shown to exert their effects by affecting association of telomere binding proteins with telomeres. Here, we use quantitative direct telomerase activity assays to evaluate the strength and mechanism of action of hydrosoluble perylene diimides (HPDIs). HPDIs contain a perylene moiety and different numbers of positively charged side chains. Side chain features vary with regard to number and distances of the charges. IC50 values of HPDIs were in the low micromolar (0.5-5 mu M) range depending on the number and features of the side chains. HPDIs having four side chains emerged as the best compounds of this series. Analysis of primer elongation products demonstrated that at low HPDI concentrations, telomerase inhibition involved formation of telomeric G-quadruplex structures, which inhibited further elongation by telomerase. At high HPDI concentrations, telomerase inhibition occurred independently of G-quadruplex formation of the substrate. The mechanism of action of HPDIs and their specific binding to G-quadruplex DNA was supported by PAGE analysis, CD spectroscopy and ESI-MS. Finally, competition Telospot experiments with duplex DNA indicated specific binding of HPDIs to the single-stranded telomeric substrates over double stranded DNA, a result supported by competitive ESI-MS. Altogether, our results indicate that HPDIs act by stabilizing G-quadruplex structures in single-stranded telomeric DNA, which in turn prevents repeat addition processivity of telomerase. (C) 2011 Elsevier Masson SAS. All rights reserved.

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5'-UUAGGG-3' repeats near their 3'-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5'-UUAGGG-3' repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres--TPP1 and the protein that tethers TPP1 to the complex, TIN2--results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.

Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of these molecules are present in the clinical setting, it is important to establish the validity of this assertion. To analyze the effects of these compounds, we used a direct assay with telomerase-enriched extracts. The comparison of potent ligands from various chemical families revealed important differences in terms of effects on telomerase initiation and processivity. Although most quadruplex ligands may lock a quadruplex-prone sequence into a quadruplex structure that inhibits the initiation of elongation by telomerase, the analysis of telomerase-elongation steps revealed that only a few molecules interfered with the processivity of telomerase (i.e., inhibit elongation once one or more repeats have been incorporated). The demonstration that these molecules are actually more effective inhibitors of telomeric DNA amplification than extension by telomerase contributes to the already growing suspicion that quadruplex ligands are not simple telomerase inhibitors but, rather, constitute a different class of biologically active molecules. We also demonstrate that the popular telomeric repeat amplification protocol is completely inappropriate for the determination of telomerase inhibition by quadruplex ligands, even when PCR controls are included. As a consequence, the inhibitory effect of many quadruplex ligands has been overestimated.

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays an essential role in immunity, neuronal function, and aging through catalysis of the rate-limiting step in the kynurenine pathway of tryptophan metabolism. Many IDO1 inhibitors with different chemotypes have been developed, mainly targeted for use in anti-cancer immunotherapy. Lead optimization of direct heme iron-binding inhibitors has proven difficult due to the remarkable selectivity and sensitivity of the heme-ligand interactions. Here, we present experimental data for a set of closely related small azole compounds with more than 4 orders of magnitude differences in their inhibitory activities, ranging from millimolar to nanomolar levels. We investigate and rationalize their activities based on structural data, molecular dynamics simulations, and density functional theory calculations. Our results not only expand the presently known four confirmed chemotypes of sub-micromolar heme binding IDO1 inhibitors by two additional scaffolds but also provide a model to predict the activities of novel scaffolds.

The activity of CAR-T cells is reversibly halted with a small-molecule drug. Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CAR-T-cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3 zeta- and CD28-containing endodomain signaling chain. We tested STOP-CAR-T cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CAR-T cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CAR-T cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies.