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Summary: Immunotherapy remains underexploited in acute myeloid leukemia (AML) compared to other hematological malignancies. Currently, gemtuzumab ozogamicin is the only therapeutic antibody approved for this disease. Here, to identify potential targets for immunotherapeutic intervention, we analyze the surface proteome of 100 genetically diverse primary human AML specimens for the identification of cell surface proteins and conduct single-cell transcriptome analyses on a subset of these specimens to assess antigen expression at the sub-population level. Through this comprehensive effort, we successfully identify numerous antigens and markers preferentially expressed by primitive AML cells. Many identified antigens are targeted by therapeutic antibodies currently under clinical evaluation for various cancer types, highlighting the potential therapeutic value of the approach. Importantly, this initiative uncovers AML heterogeneity at the surfaceome level, identifies several antigens and potential primitive cell markers characterizing AML subgroups, and positions immunotherapy as a promising approach to target AML subgroup specificities.

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/β. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

DDX3X, a mammalian RNA helicase, modulates herpes simplex virus 1 nuclear egress through cooperating with the viral nuclear egress machinery and the pUS3 viral kinase.

Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using Mapk12/Mapk13-deficient (p38γ/δKO) mice, which show low levels of TPL2, the kinase upstream of MKK1–ERK1/2 in myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here, we generated a Mapk12D171A/D171A/Mapk13−/− (p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to lipopolysaccharide (LPS)-induced septic shock and Candida albicans infection than wild-type (WT) mice. Gene expression analyses in LPS-activated wild-type and p38γ/δKIKO macrophages revealed that p38γ/p38δ-regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and in vitro kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of Nos2 and Il1b mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.

Mitotic exit requires the dephosphorylation of many proteins whose phosphorylation was needed for mitosis. Protein phosphatase 2A with its B55 regulatory subunit (PP2A-B55) promotes this transition. However, the events and substrates that it regulates are incompletely understood. We used proteomic approaches in Drosophila to identify proteins that interact with and are dephosphorylated by PP2A-B55. Among several candidates, we identified emerin (otefin in Drosophila). Emerin resides in the inner nuclear membrane and interacts with the DNA-binding protein barrier-to-autointegration factor (BAF) via a LEM domain. We found that the phosphorylation of emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, lamin and additional emerin in mitosis. We show that dephosphorylation of emerin at these sites by PP2A-B55 determines the timing of nuclear envelope reformation. Genetic experiments indicate that this regulation is required during embryonic development. Phosphoregulation of the emerin–BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely conserved across species.

Otoliths of the fish’s inner ear serve as a natural chronological recorder because of their continuous formation marked by daily, monthly, and annual increments. Despite their importance, the comprehensive protein content of otoliths remains not fully identified. Using the label-free shotgun proteomics method with one-dimensional liquid chromatography coupled to electrospray ionization-orbitrap tandem mass spectrometry, we quantified a broad range of proteins, with individual otoliths containing between 1341 and 1839 proteins. The identified proteins could potentially serve as a blueprint for fish growth from embryo to adult. We quantified eleven heat-shock proteins (HSPs) in both sexes and several proteins impacted by endocrine disruptors, indicating the otolith’s capacity to reflect environmental stress, potentially linked to climate change effects and altering of hormonal and neuroendocrine functions. Our bioinformatic ontology analysis confirmed the presence of proteins critical for various biological processes, including structural and enzymatic proteins. Protein–protein interaction (PPI) mapping also identified key interactions between the identified proteins. These findings significantly advance our understanding of otolith proteomics, offering a solid foundation for future work. Most of the identified proteins deposited daily and influenced by the environment were not implicated in the biomineralization of otolith, raising the potential for the otolith proteome to recreate details of fish life history at previously unrealized levels.

High-content imaging approaches, in combination with the use of perturbing agents such as small molecules or CRISPR-driven gene editing, have widely contributed to the identification of new therapeutic compounds. Thanks to recent advances in image-analysis methods, the use of high-content screens is increasingly gaining popularity and thus accelerating the discovery of new therapeutics. However, due to the lack of fully biocompatible fluorescent markers, large-scale high-content screens are mostly performed on fixed cells, which complicates the monitoring of changes in cell physiology over time.Here we present a novel fluorescent nontoxic dye that displays intensity and staining pattern changes in response to different physiological states. With multiparametric image analysis, these unique properties allow not only for the detection of distinct phenotypic fingerprints, but also for the quantification of more traditional disease-relevant phenotypes such as apoptosis, autophagy, ER stress and more. Since the dye only gets fluorescent when incorporated into cellular membranes, it is typically used without washing steps, therefore making it ideal to include in automation workflows. In this work, we present ​​relevant data on its biocompatibility and its potential to quantitatively assess subtle cellular phenotypes. Applications such as live kinetic imaging, and live image-based morphological profiling are also discussed. The rich information this fluorescent probe provides facilitates unbiased quantitative phenotypic analysis at larger scale, and ultimately paves the way for more discoveries of new therapeutic agents.

Abstract Apoptotic exosome-like vesicles (ApoExos) are a novel type of extracellular vesicle that contribute to the propagation of inflammation at sites of vascular injury when released by dying cells. ApoExos are characterized by the presence of the C-terminal perlecan LG3 fragment and 20S proteasome, and they are produced downstream of caspase-3 activation. In the present study, we assessed the relative roles of autophagy and caspase-3-mediated pathways in controlling the biogenesis and secretion of immunogenic ApoExos. Using electron microscopy and confocal immunofluorescence microscopy in serum-starved endothelial cells, we identified large autolysosomes resulting from the fusion of lysosomes, multivesicular bodies, and autophagosomes as a site of ApoExo biogenesis. Inhibition of autophagy with ATG7 siRNA or biochemical inhibitors (wortmannin and bafilomycin) coupled with proteomics analysis showed that autophagy regulated the processing of perlecan into LG3 and its loading onto ApoExos but was dispensable for ApoExo biogenesis. Caspase-3 activation was identified using caspase-3-deficient endothelial cells or caspase inhibitors as a pivotal regulator of fusion events between autolysosomes and the cell membrane, therefore regulating the release of immunogenic ApoExos. Collectively, these findings identified autolysosomes as a site of ApoExo biogenesis and caspase-3 as a crucial regulator of autolysosome cell membrane interactions involved in the secretion of immunogenic ApoExos.

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions. Commitment to cell division at the end of G1 phase in the budding yeast Saccharomyces cerevisiae is strongly influenced by nutrient availability. This study identifies a micro-protein that promotes G1/S transcription activation and cell cycle entry in yeast under nitrogen-limited conditions.