Many disease pathologies can be understood through the elucidation of localized biomolecular networks, or microenvironments. To this end, enzymatic proximity labeling platforms are broadly applied ...for mapping the wider spatial relationships in subcellular architectures. However, technologies that can map microenvironments with higher precision have long been sought. Here, we describe a microenvironment-mapping platform that exploits photocatalytic carbene generation to selectively identify protein-protein interactions on cell membranes, an approach we term MicroMap (μMap). By using a photocatalyst-antibody conjugate to spatially localize carbene generation, we demonstrate selective labeling of antibody binding targets and their microenvironment protein neighbors. This technique identified the constituent proteins of the programmed-death ligand 1 (PD-L1) microenvironment in live lymphocytes and selectively labeled within an immunosynaptic junction.
Dietary lipid composition has been shown to impact brain morphology, brain development, and neurologic function. However, how diet uniquely regulates brain lipid homeostasis compared with lipid ...homeostasis in peripheral tissues remains largely uncharacterized. To evaluate the lipid response to dietary changes in the brain, we assessed actively translating mRNAs in astrocytes and neurons across multiple diets. From this data, ethanolamine phosphate phospholyase (Etnppl) was identified as an astrocyte-specific fasting-induced gene. Etnppl catabolizes phosphoethanolamine (PEtN), a prominent headgroup precursor in phosphatidylethanolamine (PE) also found in other classes of neurologically relevant lipid species. Altered Etnppl expression has also previously been associated with humans with mood disorders. We evaluated the relevance of Etnppl in maintaining brain lipid homeostasis by characterizing Etnppl across development and in coregulation with PEtN-relevant genes, as well as determining the impact to the brain lipidome after Etnppl loss. We found that Etnppl expression dramatically increased during a critical window of early brain development in mice and was also induced by glucocorticoids. Using a constitutive knockout of Etnppl (EtnpplKO), we did not observe robust changes in expression of PEtN-related genes. However, loss of Etnppl altered the phospholipid profile in the brain, resulting in increased total abundance of PE and in polyunsaturated fatty acids within PE and phosphatidylcholine species in the brain. Together, these data suggest that brain phospholipids are regulated by the phospholyase action of the enzyme Etnppl, which is induced by dietary fasting in astrocytes.
The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, ...and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naïve T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection.
The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in ...high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.
The metabolic state of the brain can greatly impact neurologic function. Evidence of this includes the therapeutic benefit of a ketogenic diet in neurologic diseases, including epilepsy. However, ...brain lipid bioenergetics remain largely uncharacterized. The existence, capacity, and relevance of mitochondrial fatty acid β-oxidation (FAO) in the brain are highly controversial, with few genetic tools available to evaluate the question. We have provided evidence for the capacity of brain FAO using a pan-brain-specific conditional knockout (KO) mouse incapable of FAO due to the loss of carnitine palmitoyltransferase 2, the product of an obligate gene for FAO (CPT2
B−/−
). Loss of central nervous system (CNS) FAO did not result in gross neuroanatomical changes or systemic differences in metabolism. Loss of CPT2 in the brain did not result in robustly impaired behavior. We demonstrate by unbiased and targeted metabolomics that the mammalian brain oxidizes a substantial quantity of long-chain fatty acids in vitro and in vivo. Loss of CNS FAO results in robust accumulation of long-chain acylcarnitines in the brain, suggesting that the mammalian brain mobilizes fatty acids for their oxidation, irrespective of diet or metabolic state. Together, these data demonstrate that the mammalian brain oxidizes fatty acids under normal circumstances with little influence from or on peripheral tissues.
The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and ...binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish μMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data.
Histone deacetylase (HDAC) inhibitors (HDACis) have been widely tested in clinical trials for their ability to reverse HIV latency but have yielded only limited success. One HDACi, suberoylanilide ...hydroxamic acid (SAHA), exhibits off-target effects on host gene expression predicted to interfere with induction of HIV transcription. Romidepsin (RMD) has higher potency and specificity for class I HDACs implicated in maintaining HIV provirus in the latent state. More robust HIV reactivation has indeed been achieved with RMD use ex vivo than with SAHA; however, reduction of viral reservoir size has not been observed in clinical trials. Therefore, using RNA-Seq, we sought to compare the effects of SAHA and RMD on gene expression in primary CD4+ T cells. Among the genes whose expression was modulated by both HDACi agents, we identified genes previously implicated in HIV latency. Two genes, SMARCB1 and PARP1, whose modulation by SAHA and RMD is predicted to inhibit HIV reactivation, were evaluated in the major maturation subsets of CD4+ T cells and were consistently either up- or down-regulated by both HDACi compounds. Our results indicate that despite having different potencies and HDAC specificities, SAHA and RMD modulate an overlapping set of genes, implicated in HIV latency regulation. Some of these genes merit exploration as additional targets to improve the therapeutic outcomes of “shock and kill” strategies. The overall complexity of HDACi-induced responses among host genes with predicted stimulatory or inhibitory effects on HIV expression likely contributes to differential HDACi potencies and dictates the outcome of HIV reactivation.
The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated ...by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.
Human immunodeficiency virus (HIV) infection continues to promote neurocognitive impairment, mood disorders, and brain atrophy, even in the modern era of viral suppression. Brain lipids are ...vulnerable to HIV-associated energetic strain and may contribute to HIV-associated neurologic dysfunction due to alterations in lipid breakdown and structural lipid composition. HIV neuropathology is region dependent, yet there has not been comprehensive characterization of the spatial heterogeneity of brain lipids during infection that possibly impacts neurologic function. To address this gap, we evaluated the spatial lipid distribution using matrix laser desorption/ionization imaging mass spectrometry (MALDI-IMS) across four brain regions (parietal cortex, midbrain, thalamus, and temporal cortex), as well as the kidney for a peripheral tissue control, in a simian immunodeficiency virus (SIV)-infected rhesus macaque treated with a course of antiretroviral therapies (ARTs). We assessed lipids indicative of fat breakdown acylcarnitines (CARs) and critical structural lipids phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) across fatty acid chain lengths and degrees of unsaturation. CARs with very long-chain, polyunsaturated fatty acids (PUFAs) were more abundant across all brain regions than shorter chain, saturated, or monounsaturated species. We observed distinct brain lipid distribution patterns for the CARs and PCs. However, no clear expression patterns emerged for PEs. Surprisingly, the kidney was nearly devoid of ions corresponding to PUFAs common in brain. PEs and PCs with PUFAs had little intensity and less density than other species, and only one CAR species was observed in kidney at high intensity. Overall, our study demonstrates the stark variation in structural phospholipids and lipid-energetic intermediates present in the virally suppressed SIV-macaque brain. These findings may be useful for identifying regional vulnerabilities to damage due to brain lipid changes in people with HIV.
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