Total Synthesis and Structural Revision of Lucentamycin A

Lucentamycin A is a marine-derived peptide natural product harboring a unique 4-ethylidene-3-methylproline (Emp) subunit. The proposed structure of lucentamycin A and the core Emp residue have recently been called into question through synthesis. Here, we report the first total synthesis of lucentamycin A, which confirms that the ethylidene substituent in Emp bears an <i>E</i> geometry, in contrast to the originally assigned <i>Z</i> configuration. Synthesis of the desired (<i>E</i>)-Emp subunit required the implementation of a novel strategy starting from Garner’s aldehyde

Total Synthesis and Structural Revision of Lucentamycin A

Lucentamycin A is a marine-derived peptide natural product harboring a unique 4-ethylidene-3-methylproline (Emp) subunit. The proposed structure of lucentamycin A and the core Emp residue have recently been called into question through synthesis. Here, we report the first total synthesis of lucentamycin A, which confirms that the ethylidene substituent in Emp bears an <i>E</i> geometry, in contrast to the originally assigned <i>Z</i> configuration. Synthesis of the desired (<i>E</i>)-Emp subunit required the implementation of a novel strategy starting from Garner’s aldehyde

Deregulation of KSHV latency conformation by ER-stress and caspase-dependent RAD21-cleavage

<div><p>Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a human gammaherpesvirus recognized as the principal causative agent of KS and primary effusion lymphoma (PEL). KSHV establishes persistent latent infection in B lymphocytes where viral gene expression is restricted, in part, by a cohesin-dependent chromosome conformation. Here, we show that endoplasmic reticulum (ER) stress induces a rapid, caspase-dependent cleavage of cohesin subunit RAD21. ER stress-induced cleavage of RAD21 correlated with a rapid and strong viral lytic transcriptional activation. This effect was observed in several KSHV positive PEL cells, but not in other B-cells or non-B-cell models of KSHV latency. The cleaved-RAD21 does not dissociate from viral genomes, nor disassemble from other components of the cohesin complex. However, RAD21 cleavage correlated with the disruption of the latency genome conformation as revealed by chromosome conformation capture (3C). Ectopic expression of C-terminal RAD21 cleaved form could partially induce KSHV lytic genes transcription in BCBLI cells, suggesting that ER-stress induced RAD21 cleavage was sufficient to induce KSHV reactivation from latency in PEL cells. Taken together our results reveal a novel aspect for control and maintenance of KSHV genome latency conformation mediated by stress-induced RAD21 cleavage. Our studies also suggest that RAD21 cleavage may be a general regulatory mechanism for rapid alteration of cellular chromosome conformation and cohesin-dependent transcription regulation.</p></div

Effect of the expression of RAD21 C- terminal (aa 280–631) on KSHV reactivation and apoptosis.

<p><b>(A)</b> BCBL1 cells were transiently transfected with the GFP-tagged mammalian expression vector encoding the cleavage product hRAD21 C-terminal (aa 280 to 631). Empty vector used as a control. At 72 h post-transfection, cells lysates were subjected to immunoprecipitation with IgG or anti-GFP antibody followed by Western blot analysis with GFP antibody. <b>(B)</b> BCBL1 cells treated as in (A) were subjected to RNA extraction and RT-qPCR for latent transcript LANA, lytic transcripts ORF50 and PAN. (C) Same as in panel B, except RT-qPCR for cellular genes PHGDH and myc tested as cellular negative controls (left panel), or the 3’ end of RAD21 (ectopic transcript), or the 5’ end of RAD21 (endogenous transcript). * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Effect of ER stress inducers on the epigenomic programming of KSHV regulatory control regions.

<p><b>(A)</b> Schematic of KSHV lytic control region, latency control region and terminal repeats region (TR) with primer positions a-h used for ChIP assays. Red triangles represent CTCF binding sites. <b>(B-E)</b> BCBL1 cells treated for 3 hrs with DMSO or 75 ug/ml Puro were assayed by ChIP for IgG or <b>(B</b>) RAD21, <b>(C)</b> CTCF, <b>(D)</b> RNA Polymerase II phosphoS5 (RNAPII-S5) and <b>(E)</b> RNA Polymerase II phosphoS2 (RNAPII-S2). <b>(F)</b> BCBL1 cells were treated with puromycin for 6 hr, processed for IP with antibody to IgG, SMC1 or RAD21 and then assayed by Western blot with antibody to RAD21, SMC3 or SMC1. <b>(G)</b> BCBL1 cells were treated with puromycin for 3 and 6 hr, subcellular fractionation was performed, and cytoplasmic, nucleoplasmic and chromatin fractions were analyzed by Western blotting. GAPDH (a cytoplasmic protein), and chromatin bound histone H3 protein were used as controls for subcellular fractionation.</p

ER stress inducers promote RAD21 cleavage and KSHV lytic reactivation in BCBLI cells.

<p><b>(A)</b> Immunoblotting of RAD21, PARP, SMC1, SMC3, ORF45 and actin in BCBLI cells exposed to 5mM DTT or 75ug/ml Puromycin (Puro) for 6 hours. <b>(B-C)</b> RT-qPCR for latent transcripts (LANA, ORF71, ORF72), lytic transcripts (ORF45-50, PAN), and RAD21 transcript relative to cellular actin in BCBLI cells treated as in (A). The data are expressed as fold change of the treated versus untreated (DMSO) cells. <b>(D)</b> Immunoblotting of RAD21, PARP, BiP and actin in BCBLI cells exposed to 0.1 ug/ml of SubAB for 1, 3, 6, 9 and 24 hours. <b>(E)</b> RT-qPCR for latent transcript LANA, lytic transcripts (ORF45-50, PAN), and RAD21 transcript relative to cellular actin in BCBLI cells treated as in (D). The data are expressed as fold change of the treated versus untreated (time 0h) cells. <b>(F)</b> Immunoblotting of ORF45, ORF50, and actin in BCBLI cells exposed to 5mM DTT or 75ug/ml Puromycin (Puro) or 3mM sodium butyrate (SB) for 6 hours.</p

RAD21 cleavage promotes DNA-loop disruption between KSHV latent and lytic control regions.

<p><b>(A)</b> Schematic of KSHV regulatory regions and primers used for 3C experiment. Anchor primer at latency control region (128,264) indicated by asterisk (*) and acceptor sites in lytic control region (69163), and others regions are indicated. Red triangles represent CTCF binding sites. <b>(B-C)</b> Chromosome conformation capture <b>(</b>3C) assay in BCBL1 cells treated for 3–6 hrs with DMSO or 5mM DTT (B) or 75 ug/ml Puro (C) using anchor and acceptor primers, as indicated in (A). <b>(D)</b> Chromosome conformation capture <b>(</b>3C) assay in BCBL1 cells treated for 3 hrs with 75 ug/ml Puro in the presence or in the absence of 20 uM z-VAD-FMK using anchor and acceptor primers, as indicated in (A). * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Induction of RAD21 degradation and KSHV lytic cycle by protein synthesis inhibitors in BCBLI cells.

<p><b>(A)</b> Immunoblotting of RAD21 in KSHV latently infected BCBLI cells exposed to different antibiotics. BCBLI cells were incubated with 10ug/ml Emetine, 75ug/ml Puromycin, 0.2 ug/ml Pactamycin, 10ug/ml Anisomycin or 100 ug/ml cycloheximide for 0, 2, 4, 6 and 8 hours. <b>(B-F)</b> RT-qPCR for ORF45-50, LANA and RAD21 transcripts relative to cellular 18S in BCBLI cells treated for 0–8 hrs with Emetine (EME), Puromycin (Puro), Pactamycin (Pact), Anisomycin (Anis) or Cycloheximide (CHX) as described in (A).</p

Model of RAD21 cleavage and KSHV lytic induction by ER stress inducers.

<p>ER stress leads to activation of the UPR pathway and apoptotic caspase cascade. Apoptosis triggers the degradation of cellular RAD21, one of the main proteins responsible for the maintenance of KSHV episome conformation during latency. The cleavage of RAD21 promotes the disruption of the KSHV DNA loop between the latency and lytic regulatory control regions and consequently the switch to KSHV lytic phase.</p

ER stress induces KSHV lytic replication in BCBL1 cells.

<p><b>(A)</b> Immunoblotting of RAD21, PARP and actin in BJAB, SLK-BAC16 and BCBL1 cells exposed to 5mM DTT or 75ug/ml Puromycin (Puro) for 6 hours. <b>(B)</b> RT-qPCR for latent transcript LANA, lytic transcripts ORF50 and PAN, and RAD21 transcript relative to cellular actin in SLK-BAC16 and BCBL1 cells treated with DMSO, 5mM DTT or 75ug/ml Puromycin (Puro) for 6 hours. The data are expressed as fold change of the treated versus untreated (DMSO) cells. <b>(C)</b> Schematic representation of drug treatments in BCBL1 cells. Cells were treated with a 6-hour pulse of DMSO or 5mM DTT or 75ug/ml Puromycin (Puro) and then cultured in fresh media lacking drugs for the remaining 24 hours or 96 hours. <b>(D)</b> At 6 and 24 hours after drug treatment as described in (C), the level of viral DNA was evaluated by real-time qPCR for ORF50 DNA relative to cellular actin. The data are expressed as fold change of the treated versus untreated (DMSO) cells. <b>(E)</b> Virion DNA was isolated from cell supernatants at 96 h post-treatment and wash-out and assayed by qPCR for ORF50 DNA. Sodium butyrate (SB) treated cell supernatant was used as positive control. The data are expressed as fold change of the treated versus untreated (DMSO) cells.</p