HMBPP Analog Prodrugs Bypass Energy-Dependent Uptake To Promote Efficient BTN3A1-Mediated Malignant Cell Lysis by Vγ9Vδ2 T Lymphocyte Effectors.

Vγ9Vδ2 effector T cells lyse cells in response to phosphorus-containing small molecules, providing primates a unique route to remove infected or malignant cells. Yet, the triggering mechanisms remain ill defined. We examined lysis mediated by human Vγ9Vδ2 effector T cells in response to the naturally occurring (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) or a synthetic cell-permeable prodrug, bis (pivaloyloxymethyl) (E)-4-hydroxy-3-methyl-but-2-enyl phosphonate. CD27(+)/CD45RA(-) Th1-like effector cells killed K562 target cells through a mechanism that could be enhanced by either compound or TCR Ab and blocked by Src inhibition or butyrophilin 3 isoform A1 (BTN3A1) disruption. Pretreatment at 4 °: C decreased HMBPP-induced lysis but did not reduce lysis induced by bis (pivaloyloxymethyl) (E)-4-hydroxy-3-methyl-but-2-enyl phosphonate. Together, our results show that internalization of HMBPP into target cells is required for BTN3A1-dependent lysis by Vγ9Vδ2 effector T cells. The enhanced activity of the prodrug analog is due to its ability to bypass the pathways required for entry of HMBPP. These findings support an inside-out model of T cell triggering driven by small-molecule induction of BTN3A1.

Evaluation of a 7-Methoxycoumarin-3-carboxylic Acid Ester Derivative as a Fluorescent, Cell-Cleavable, Phosphonate Protecting Group.

Cell-cleavable protecting groups often enhance cellular delivery of species that are charged at physiological pH. Although several phosphonate protecting groups have achieved clinical success, it remains difficult to use these prodrugs in live cells to clarify biological mechanisms. Here, we present a strategy that uses a 7-methoxycoumarin-3-carboxylic acid ester as a fluorescent protecting group. This strategy was applied to synthesis of an (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) analogue to assess cellular uptake and human Vγ9Vδ2 T cell activation. The fluorescent ester displayed low cellular toxicity (IC50 >100 µm) and strong T cell activation (EC50 =0.018 µm) relative to the unprotected anion (EC50 =23 µm). The coumarin-derived analogue allowed no-wash analysis of biological deprotection, which revealed rapid internalization of the prodrug. These results demonstrate that fluorescent groups can be applied both as functional drug delivery tools and useful biological probes of drug uptake.

Minor splicing snRNAs are enriched in the developing mouse CNS and are crucial for survival of differentiating retinal neurons.

In eukaryotes, gene expression requires splicing, which starts with the identification of exon-intron boundaries by the small, nuclear RNA (snRNAs) of the spliceosome, aided by associated proteins. In the mammalian genome, <1% of introns lack canonical exon-intron boundary sequences and cannot be spliced by the canonical splicing machinery. These introns are spliced by the minor spliceosome, consisting of unique snRNAs (U11, U12, U4atac, and U6atac). The importance of the minor spliceosome is underscored by the disease microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1), which is caused by mutation in U4atac. Thus, it is important to understand the expression and function of the minor spliceosome and its targets in mammalian development, for which we used the mouse as our model. Here, we report enrichment of the minor snRNAs in the developing head/central nervous system (CNS) between E9.5 and E12.5, along with enrichment of these snRNAs in differentiating retinal neurons. Moreover, dynamic expression kinetics of minor intron-containing genes (MIGs) was observed across retinal development. DAVID analysis of MIGs that were cotranscriptionally upregulated embryonically revealed enrichment for RNA metabolism and cell cycle regulation. In contrast, MIGs that were cotranscriptionally upregulated postnatally revealed enrichment for protein localization/transport, vesicle-mediated transport, and calcium transport. Finally, we used U12 morpholino to inactivate the minor spliceosome in the postnatal retina, which resulted in apoptosis of differentiating retinal neurons. Taken together, our data suggest that the minor spliceosome may have distinct functions in embryonic versus postnatal development. Importantly, we show that the minor spliceosome is crucial for the survival of terminally differentiating retinal neurons.

Replication-dependent histone genes are actively transcribed in differentiating and aging retinal neurons.

In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.