A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.

The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular functions. Here, we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7,000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of protein modules in centriole duplication, ciliogenesis, and centriolar satellite biogenesis and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.

A proximity-dependent biotinylation map of a human cell.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

MAPL loss dysregulates bile and liver metabolism in mice.

Mitochondrial and peroxisomal anchored protein ligase (MAPL) is a dual ubiquitin and small ubiquitin-like modifier (SUMO) ligase with roles in mitochondrial quality control, cell death and inflammation in cultured cells. Here, we show that MAPL function in the organismal context converges on metabolic control, as knockout mice are viable, insulin-sensitive, and protected from diet-induced obesity. MAPL loss leads to liver-specific activation of the integrated stress response, inducing secretion of stress hormone FGF21. MAPL knockout mice develop fully penetrant spontaneous hepatocellular carcinoma. Mechanistically, the peroxisomal bile acid transporter ABCD3 is a primary MAPL interacting partner and SUMOylated in a MAPL-dependent manner. MAPL knockout leads to increased bile acid production coupled with defective regulatory feedback in liver in vivo and in isolated primary hepatocytes, suggesting cell-autonomous function. Together, our findings establish MAPL function as a regulator of bile acid synthesis whose loss leads to the disruption of bile acid feedback mechanisms. The consequences of MAPL loss in liver, along with evidence of tumor suppression through regulation of cell survival pathways, ultimately lead to hepatocellular carcinogenesis.

Analyses of combined Merkel cell carcinomas with neuroblastic components suggests that loss of T antigen expression in Merkel cell carcinoma may result in cell cycle arrest and neuroblastic transdifferentiation.

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by genomic integration of the Merkel cell polyomavirus (MCPyV). MCPyV-negative cases often present as combined MCCs, which represent a distinctive subset of tumors characterized by association of an MCC with a second tumor component, mostly squamous cell carcinoma. Up to now, only exceptional cases of combined MCC with neuroblastic differentiation have been reported. Herein we describe two additional combined MCCs with neuroblastic differentiation and provide comprehensive morphologic, immunohistochemical, transcriptomic, genetic and epigenetic characterization of these tumors, which both arose in elderly men and appeared as an isolated inguinal adenopathy. Microscopic examination revealed biphasic tumors combining a poorly differentiated high-grade carcinoma with a poorly differentiated neuroblastic component lacking signs of proliferation. Immunohistochemical investigation revealed keratin 20 and MCPyV T antigen (TA) in the MCC parts, while neuroblastic differentiation was confirmed in the other component in both cases. A clonal relation of the two components can be deduced from 20 and 14 shared acquired point mutations detected by whole exome analysis in both combined tumors, respectively. Spatial transcriptomics demonstrated a lower expression of stem cell marker genes such as SOX2 and MCM2 in the neuroblastic component. Interestingly, although the neuroblastic part lacked TA expression, the same genomic MCPyV integration and the same large T-truncating mutations were observed in both tumor parts. Given that neuronal transdifferentiation upon TA repression has been reported for MCC cell lines, the most likely scenario for the two combined MCC/neuroblastic tumors is that neuroblastic transdifferentiation resulted from loss of TA expression in a subset of MCC cells. Indeed, DNA methylation profiling suggests an MCC-typical cellular origin for the combined MCC/neuroblastomas. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

LLGL2 rescues nutrient stress by promoting leucine uptake in ER+ breast cancer.

Drosophila Lgl and its mammalian homologues, LLGL1 and LLGL2, are scaffolding proteins that regulate the establishment of apical-basal polarity in epithelial cells1,2. Whereas Lgl functions as a tumour suppressor in Drosophila1, the roles of mammalian LLGL1 and LLGL2 in cancer are unclear. The majority (about 75%) of breast cancers express oestrogen receptors (ERs)3, and patients with these tumours receive endocrine treatment4. However, the development of resistance to endocrine therapy and metastatic progression are leading causes of death for patients with ER+ disease4. Here we report that, unlike LLGL1, LLGL2 is overexpressed in ER+ breast cancer and promotes cell proliferation under nutrient stress. LLGL2 regulates cell surface levels of a leucine transporter, SLC7A5, by forming a trimeric complex with SLC7A5 and a regulator of membrane fusion, YKT6, to promote leucine uptake and cell proliferation. The oestrogen receptor targets LLGL2 expression. Resistance to endocrine treatment in breast cancer cells was associated with SLC7A5- and LLGL2-dependent adaption to nutrient stress. SLC7A5 was necessary and sufficient to confer resistance to tamoxifen treatment, identifying SLC7A5 as a potential therapeutic target for overcoming resistance to endocrine treatments in breast cancer. Thus, LLGL2 functions as a promoter of tumour growth and not as a tumour suppressor in ER+ breast cancer. Beyond breast cancer, adaptation to nutrient stress is critically important5, and our findings identify an unexpected role for LLGL2 in this process.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.

The in vivo Interaction Landscape of Histones H3.1 and H3.3.

Chromatin structure, transcription, DNA replication, and repair are regulated via locus-specific incorporation of histone variants and posttranslational modifications that guide effector chromatin-binding proteins. Here we report unbiased, quantitative interactomes for the replication-coupled (H3.1) and replication-independent (H3.3) histone H3 variants based on BioID proximity labeling, which allows interactions in intact, living cells to be detected. Along with a significant proportion of previously reported interactions detected by affinity purification followed by mass spectrometry, three quarters of the 608 histone-associated proteins that we identified are new, uncharacterized histone associations. The data reveal important biological nuances not captured by traditional biochemical means. For example, we found that the chromatin assembly factor-1 histone chaperone not only deposits the replication-coupled H3.1 histone variant during S-phase but also associates with H3.3 throughout the cell cycle in vivo. We also identified other variant-specific associations, such as with transcription factors, chromatin regulators, and with the mitotic machinery. Our proximity-based analysis is thus a rich resource that extends the H3 interactome and reveals new sets of variant-specific associations.

Spatial and proteomic profiling reveals centrosome-independent features of centriolar satellites.

Centriolar satellites are small electron-dense granules that cluster in the vicinity of centrosomes. Satellites have been implicated in multiple critical cellular functions including centriole duplication, centrosome maturation, and ciliogenesis, but their precise composition and assembly properties have remained poorly explored. Here, we perform in vivo proximity-dependent biotin identification (BioID) on 22 human satellite proteins, to identify 2,113 high-confidence interactions among 660 unique polypeptides. Mining this network, we validate six additional satellite components. Analysis of the satellite interactome, combined with subdiffraction imaging, reveals the existence of multiple unique microscopically resolvable satellite populations that display distinct protein interaction profiles. We further show that loss of satellites in PCM1-depleted cells results in a dramatic change in the satellite interaction landscape. Finally, we demonstrate that satellite composition is largely unaffected by centriole depletion or disruption of microtubules, indicating that satellite assembly is centrosome-independent. Together, our work offers the first systematic spatial and proteomic profiling of human centriolar satellites and paves the way for future studies aimed at better understanding the biogenesis and function(s) of these enigmatic structures.

Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.

Oncohistone interactome profiling uncovers contrasting oncogenic mechanisms and identifies potential therapeutic targets in high grade glioma.

Histone H3 mutations at amino acids 27 (H3K27M) and 34 (H3G34R) are recurrent drivers of pediatric-type high-grade glioma (pHGG). H3K27M mutations lead to global disruption of H3K27me3 through dominant negative PRC2 inhibition, while H3G34R mutations lead to local losses of H3K36me3 through inhibition of SETD2. However, their broader oncogenic mechanisms remain unclear. We characterized the H3.1K27M, H3.3K27M and H3.3G34R interactomes, finding that H3K27M is associated with epigenetic and transcription factor changes; in contrast H3G34R removes a break on cryptic transcription, limits DNA methyltransferase access, and alters mitochondrial metabolism. All 3 mutants had altered interactions with DNA repair proteins and H3K9 methyltransferases. H3K9me3 was reduced in H3K27M-containing nucleosomes, and cis-H3K9 methylation was required for H3K27M to exert its effect on global H3K27me3. H3K9 methyltransferase inhibition was lethal to H3.1K27M, H3.3K27M and H3.3G34R pHGG cells, underscoring the importance of H3K9 methylation for oncohistone-mutant gliomas and suggesting it as an attractive therapeutic target.

ARID1a Associates with Lymphoid-Restricted Transcription Factors and Has an Essential Role in T Cell Development.

Maturation of lymphoid cells is controlled by the action of stage and lineage-restricted transcription factors working in concert with the general transcription and chromatin remodeling machinery to regulate gene expression. To better understand this functional interplay, we used Biotin Identification in human embryonic kidney cells to identify proximity interaction partners for GATA3, TCF7 (TCF1), SPI1, HLF, IKZF1, PAX5, ID1, and ID2. The proximity interaction partners shared among the lineage-restricted transcription factors included ARID1a, a BRG1-associated factor complex component. CUT&RUN analysis revealed that ARID1a shared binding with TCF7 and GATA3 at a substantial number of putative regulatory elements in mouse T cell progenitors. In support of an important function for ARID1a in lymphocyte development, deletion of Arid1a in early lymphoid progenitors in mice resulted in a pronounced developmental arrest in early T cell development with a reduction of CD4+CD8+ cells and a 20-fold reduction in thymic cellularity. Exploring gene expression patterns in DN3 cells from Wt and Arid1a-deficient mice suggested that the developmental block resided in the DN3a to DN3b transition, indicating a deficiency in ß-selection. Our work highlights the critical importance of functional interactions between stage and lineage-restricted factors and the basic transcription machinery during lymphocyte differentiation.

Alternative proteins are functional regulators in cell reprogramming by PKA activation.

It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

BioID Performed on Golgi Enriched Fractions Identify C10orf76 as a GBF1 Binding Protein Essential for Golgi Maintenance and Secretion.

The Golgi-specific Brefeldin-A resistance factor 1 (GBF1) is the only large GEF that regulates Arf activation at the cis-Golgi and is actively recruited to membranes on an increase in Arf-GDP. Recent studies have revealed that GBF1 recruitment requires one or more heat-labile and protease-sensitive protein factor(s) (Quilty et al., 2018, J. Cell Science, 132). Proximity-dependent biotinylation (BioID) and mass spectrometry from enriched Golgi fractions identified GBF1 proximal proteins that may regulate its recruitment. Knockdown studies revealed C10orf76 to be involved in Golgi maintenance. We find that C10orf76 interacts with GBF1 and rapidly cycles on and off GBF1-positive Golgi structures. More importantly, its depletion causes Golgi fragmentation, alters GBF1 recruitment, and impairs secretion. Homologs were identified in most species, suggesting its presence in the last eukaryotic common ancestor.

Deficiency of the autophagy gene ATG16L1 induces insulin resistance through KLHL9/KLHL13/CUL3-mediated IRS1 degradation.

Connections between deficient autophagy and insulin resistance have emerged, however, the mechanism through which reduced autophagy impairs insulin-signaling remains unknown. We examined mouse embryonic fibroblasts lacking Atg16l1 (ATG16L1 KO mouse embryonic fibroblasts (MEFs)), an essential autophagy gene, and observed deficient insulin and insulin-like growth factor-1 signaling. ATG16L1 KO MEFs displayed reduced protein content of insulin receptor substrate-1 (IRS1), pivotal to insulin signaling, whereas IRS1myc overexpression recovered downstream insulin signaling. Endogenous IRS1 protein content and insulin signaling were restored in ATG16L1 KO mouse embryonic fibroblasts (MEF) upon proteasome inhibition. Through proximity-dependent biotin identification (BioID) and co-immunoprecipitation, we found that Kelch-like proteins KLHL9 and KLHL13, which together form an E3 ubiquitin (Ub) ligase complex with cullin 3 (CUL3), are novel IRS1 interactors. Expression of Klhl9 and Klhl13 was elevated in ATG16L1 KO MEFs and siRNA-mediated knockdown of Klhl9, Klhl13, or Cul3 recovered IRS1 expression. Moreover, Klhl13 and Cul3 knockdown increased insulin signaling. Notably, adipose tissue of high-fat fed mice displayed lower Atg16l1 mRNA expression and IRS1 protein content, and adipose tissue KLHL13 and CUL3 expression positively correlated to body mass index in humans. We propose that ATG16L1 deficiency evokes insulin resistance through induction of Klhl9 and Klhl13, which, in complex with Cul3, promote proteasomal IRS1 degradation.

Global Interactomics Uncovers Extensive Organellar Targeting by Zika Virus.

Zika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. >60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection.

Global Interactome Mapping of Mitochondrial Intermembrane Space Proteases Identifies a Novel Function for HTRA2.

A number of unique proteases localize to specific sub-compartments of the mitochondria, but the functions of these enzymes are poorly defined. Here, in vivo proximity-dependent biotinylation (BioID) is used to map the interactomes of seven proteases localized to the mitochondrial intermembrane space (IMS). In total, 802 high confidence proximity interactions with 342 unique proteins are identified. While all seven proteases co-localized with the IMS markers OPA1 and CLPB, 230 of the interacting partners are unique to just one or two protease bait proteins, highlighting the ability of BioID to differentiate unique interactomes within the confined space of the IMS. Notably, high-temperature requirement peptidase 2 (HTRA2) interacts with eight of 13 components of the mitochondrial intermembrane space bridging (MIB) complex, a multiprotein assembly essential for the maintenance of mitochondrial cristae structure. Knockdown of HTRA2 disrupts cristae in HEK 293 and OCI-AML2 cells, and leads to increased intracellular levels of the MIB subunit IMMT. Using a cell-free assay it is demonstrated that HTRA2 can degrade recombinant IMMT but not two other core MIB complex subunits, SAMM50 and CHCHD3. The IMS protease interactome thus represents a rich dataset that can be mined to uncover novel IMS protease biology.