Search Results (38)

SUMOylation plays a crucial role in regulating gene expression by promoting interactions between transcription factors and corepressors. Daxx, a multifunctional scaffold protein, specifically recognizes and binds SUMOylated transcription factors through its SUMO-interacting motifs (SIMs), acting as a transcriptional corepressor. In this review, we systematically elucidate the structural basis of the interaction between Daxx and SUMO, revealing the synergistic mechanism by which Daxx SIM phosphorylation and SUMO acetylation dynamically regulate Daxx function. In promyelocytic leukemia nuclear bodies (PML NBs), phosphorylation of Daxx’s SIM enhances its binding to SUMOylated PML, leading to the sequestration and inactivation of Daxx within PML NBs. Conversely, SUMO acetylation disrupts the electrostatic interactions between SUMO and SIMs, prompting the release of Daxx from PML NBs and its translocation to the nucleoplasm, where it inhibits the activity of transcription factors such as ETS1, GR, and SMAD4. Daxx SIMs are common binding sites for the interaction between SUMOylated transcription factors and Daxx, and different SUMOylated transcription factors may compete to bind to Daxx, which cross-regulates cellular life activities. This mechanism highlights the dynamic regulation of Daxx subcellular localization and transcriptional repression by SUMO and PML NBs, providing valuable insights into understanding Daxx-mediated transcriptional repression. Full article

The promyelocytic leukemia protein (PML) and its associated nuclear bodies have recently emerged as critical regulators of embryonic stem (ES) cell identity. Despite their recognized importance, the complete spectrum of PML-mediated molecular events in ES cells remains unclear. In this report, we study how PML is shaping the proteomic and SUMO proteomic landscape in ES cells. Proteomic profiling of PML-depleted ES cells uncovered a downregulation of self-renewal factors and an upregulation of proteins associated with translation and proteasomal activity, reflecting a cellular transition from pluripotency to differentiation. Importantly, PML promotes the sumoylation of pluripotency-related factors, chromatin organizers, and cell cycle regulators. We identified SALL1 and CDCA8 as novel PML-directed sumoylation targets, both critical for ES cell maintenance. SALL1 sumoylation increases the activation of the Wnt pathway, contributing to its ability to inhibit ES cell differentiation. Similarly, CDCA8 sumoylation enhances its capacity to promote cell proliferation. Collectively, our findings demonstrate that PML regulates ES cell identity by modulating the abundance or sumoylation of key regulators involved in pluripotency and cell cycle progression. Full article

Natural aging and age-related diseases involve the acceleration of replicative aging, or senescence. Multiple proteins are known to participate in these processes, including the promyelocytic leukemia (PML) protein, which serves as a core component of nuclear-membrane-less organelles known as PML nuclear bodies (PML-NBs). In this work, morphological changes in PML-NBs and alterations in PML protein localization at the transition of primary fibroblasts to a replicative senescent state were studied by immunofluorescence. The fibroblasts were obtained from both healthy donors and donors with premature aging syndromes (ataxia-telangiectasia and Cockayne syndrome). Our data showed an increase in both the size and the number of PML-NBs, along with nuclear enlargement in senescent cells, suggesting these changes could serve as potential cellular aging markers. Bioinformatic analysis demonstrated that 30% of the proteins in the PML interactome and ~45% of the proteins in the PML-NB predicted proteome are directly associated with senescence and aging processes. These proteins are hypothesized to participate in post-translational modifications and protein sequestration within PML-NBs, thereby influencing transcription factor regulation, DNA damage response, and negative regulation of apoptosis. The findings confirm the significant role of PML-NBs in cellular aging processes and open new avenues for investigating senescence mechanisms and age-associated diseases. Full article

Ataxia Telangiectasia and Rad3-related protein (ATR) is an apical kinase of the DNA Damage Response (DDR) pathway responsible for detecting and resolving damaged DNA. Because cancer cells depend heavily on the DNA damage checkpoint for their unchecked proliferation and propagation, ATR has gained enormous popularity as a cancer therapy target in recent decades. Yet, ATR inhibitors have not been the silver bullets as anticipated, with clinical trials demonstrating toxicity and mixed efficacy. To investigate whether the toxicity and mixed efficacy of ATR inhibitors arise from their off-target effects related to ATR’s multiple roles within and outside the DDR pathway, we have analyzed recently published studies on ATR’s non-canonical roles. Recent studies have elucidated that ATR plays a wide role throughout the cell cycle that is separate from its function in the DDR. This includes maintaining nuclear membrane integrity, detecting mechanical forces, and promoting faithful chromosome segregation during mitosis. In this review, we summarize the canonical, DDR-related roles of ATR and also focus on the non-canonical, multifaceted roles of ATR throughout the cell cycle and their clinical relevance. Through this summary, we also address the need for re-assessing clinical strategies targeting ATR as a cancer therapy based on these newly discovered roles for ATR. Full article

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are core–shell-type membrane-less organelles typically found in the nucleus of mammalian somatic cells but are absent in mouse oocytes. Here, we deliberately induced the assembly of PML-NBs by injecting mRNA encoding human PML protein (hPML VI -sfGFP) into oocytes and investigated their impact on fertilization in which oocyte/embryos undergo multiple types of stresses. Following nuclear membrane breakdown, preassembled hPML VI -sfGFP mRNA-derived PML-NBs (hmdPML-NBs) persisted in the cytoplasm of oocytes, forming less-soluble debris, particularly under stress. Parthenogenetic embryos that successfully formed pronuclei were capable of removing preassembled hmdPML-NBs from the cytoplasm while forming new hmdPML-NBs in the pronucleus. These observations highlight the beneficial aspect of the PML-NB-free nucleoplasmic environment and suggest that the ability to eliminate unnecessary materials in the cytoplasm of metaphase oocytes serves as a potential indicator of the oocyte quality. Full article

The story of acute promyelocytic leukemia (APL) discovery, physiopathology, and treatment is a unique journey, transforming the most aggressive form of leukemia to the most curable. It followed an empirical route fueled by clinical breakthroughs driving major advances in biochemistry and cell biology, including the discovery of PML nuclear bodies (PML NBs) and their central role in APL physiopathology. Beyond APL, PML NBs have emerged as key players in a wide variety of biological functions, including tumor-suppression and SUMO-initiated protein degradation, underscoring their broad importance. The APL story is an example of how clinical observations led to the incremental development of the first targeted leukemia therapy. The understanding of APL pathogenesis and the basis for cure now opens new insights in the treatment of other diseases, especially other acute myeloid leukemias. Full article

The formation and function of membrane-less organelles (MLOs) is one of the main driving forces in the molecular life of the cell. These processes are based on the separation of biopolymers into phases regulated by multiple specific and nonspecific inter- and intramolecular interactions. Among the realm of MLOs, a special place is taken by the promyelocytic leukemia nuclear bodies (PML-NBs or PML bodies), which are the intranuclear compartments involved in the regulation of cellular metabolism, transcription, the maintenance of genome stability, responses to viral infection, apoptosis, and tumor suppression. According to the accepted models, specific interactions, such as SUMO/SIM, the formation of disulfide bonds, etc., play a decisive role in the biogenesis of PML bodies. In this work, a number of bioinformatics approaches were used to study proteins found in the proteome of PML bodies for their tendency for spontaneous liquid–liquid phase separation (LLPS), which is usually caused by weak nonspecific interactions. A total of 205 proteins found in PML bodies have been identified. It has been suggested that UBC9, P53, HIPK2, and SUMO1 can be considered as the scaffold proteins of PML bodies. It was shown that more than half of the proteins in the analyzed proteome are capable of spontaneous LLPS, with 85% of the analyzed proteins being intrinsically disordered proteins (IDPs) and the remaining 15% being proteins with intrinsically disordered protein regions (IDPRs). About 44% of all proteins analyzed in this study contain SUMO binding sites and can potentially be SUMOylated. These data suggest that weak nonspecific interactions play a significantly larger role in the formation and biogenesis of PML bodies than previously expected. Full article

Nuclear bodies (NBs) are dynamic structures present in eukaryotic cell nuclei. They are not bounded by membranes and are often considered biomolecular condensates, defined structurally and functionally by the localisation of core components. Nuclear architecture can be reorganised during normal cellular processes such as the cell cycle as well as in response to cellular stress. Many plant and animal viruses target their proteins to NBs, in some cases triggering their structural disruption and redistribution. Although not all such interactions have been well characterised, subversion of NBs and their functions may form a key part of the life cycle of eukaryotic viruses that require the nucleus for their replication. This review will focus on Cajal bodies (CBs) and the viruses that target them. Since CBs are dynamic structures, other NBs (principally nucleoli and promyelocytic leukaemia, PML and bodies), whose components interact with CBs, will also be considered. As well as providing important insights into key virus–host cell interactions, studies on Cajal and associated NBs may identify novel cellular targets for development of antiviral compounds. Full article

Upon viral entry, components of ND10 nuclear bodies converge with incoming DNA to repress viral expression. The infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) contains a RING-type E3 ubiquitin ligase that targets the ND10 organizer, PML, for proteasomal degradation. Consequently, ND10 components are dispersed and viral genes are activated. Previously, we reported that ICP0 E3 differentiates two similar substrates, PML isoforms I and II, and demonstrated that SUMO-interaction has profound regulatory effects on PML II degradation. In the present study, we investigated elements that regulate the PML I degradation and found that: (i) two regions of ICP0 flanking the RING redundantly facilitate the degradation of PML I; (ii) downstream of the RING, the SUMO-interaction motif located at residues 362–364 (SIM_362–364) targets the SUMOylated PML I in the same manner as that of PML II; (iii) upstream of the RING, the N-terminal residues 1–83 mediate PML I degradation regardless of its SUMOylation status or subcellular localization; (iv) the reposition of residues 1–83 to downstream of the RING does not affect its function in PML I degradation; and (v) the deletion of 1–83 allows the resurgence of PML I and reformation of ND10-like structures late in HSV-1 infection. Taken together, we identified a novel substrate recognition specific for PML I, by which ICP0 E3 enforces a continuous PML I degradation throughout the infection to prevent the ND10 reformation. Full article

In addition to its function as an intravascular lipid transporter, LDL also triggers signal transduction in endothelial cells (ECs), which, among other things, trigger immunomodulatory cascades, e.g., IL-6 upregulation. However, the molecular mechanisms of how these LDL-triggered immunological responses in ECs are realized are not fully understood. Since promyelocytic leukemia protein (PML) plays a role in promoting inflammatory processes, we examined the relationship between LDL, PML, and IL-6 in human ECs (HUVECs and EA.hy926 cells). RT-qPCR, immunoblotting, and immunofluorescence analyses showed that LDL but not HDL induced higher PML expression and higher numbers of PML-nuclear bodies (PML-NBs). Transfection of the ECs with a PML gene-encoding vector or PML-specific siRNAs demonstrated PML-regulated IL-6 and IL-8 expression and secretion after LDL exposure. Moreover, incubation with the PKC inhibitor sc-3088 or the PKC activator PMA showed that LDL-induced PKC activity leads to the upregulation of PML mRNA and PML protein. In summary, our experimental data suggest that high LDL concentrations trigger PKC activity in ECs to upregulate PML expression, which then increases production and secretion of IL-6 and IL-8. This molecular cascade represents a novel cellular signaling pathway with immunomodulatory effects in ECs in response to LDL exposure. Full article

PML nuclear bodies (PML-NBs) are dynamic macromolecular complexes that mediate intrinsic immunity against viruses of different families, including human cytomegalovirus (HCMV). Upon HCMV infection, PML-NBs target viral genomes entering the nucleus and restrict viral immediate–early gene expression by epigenetic silencing. Studies from several groups performed in human fibroblast cells have shown that the major PML-NB components PML, Daxx, Sp100 and ATRX contribute to this repression in a cooperative manner. Their role for HCMV restriction in endothelial cells, however, has not yet been characterized although infected endothelium is thought to play a crucial role for HCMV dissemination and development of vascular disease in vivo. Here, we use conditionally immortalized umbilical vein endothelial cells (HEC-LTT) as a cell culture model to elucidate the impact of PML-NB proteins on lytic HCMV infection. Depletion of individual PML-NB proteins by lentiviral transduction showed a particularly strong antiviral effect of PML in HEC-LTT, compared to human fibroblasts. A closer characterization of this antiviral function revealed that PML may not only effectively inhibit HCMV immediate-early gene expression but also act at later steps of the viral replication cycle. At contrast, we surprisingly noted an antiviral behavior of Daxx in complementary approaches: Depletion of Daxx resulted in decreased viral gene expression, while overexpression of Daxx promoted HCMV infection. In summary, our data demonstrate a cell type-specific effect of PML-NB components on lytic HCMV infection and suggest an important role of PML in the inhibition of HCMV dissemination through infected endothelial cells. Full article

By forming specific functional entities, nuclear biomolecular condensates play an important function in guiding biological processes. PML biomolecular condensates, also known as PML nuclear bodies (NBs), are macro-molecular sub-nuclear organelles involved in central biological processes, including anti-viral response and cell fate control upon genotoxic stress. PML condensate formation is stimulated upon cellular stress, and relies on protein–protein interactions establishing a PML protein meshwork capable of recruiting the tumor suppressor p53, along with numerous modifiers of p53, thus balancing p53 posttranslational modifications and activity. This stress-regulated process appears to be controlled by liquid–liquid phase separation (LLPS), which may facilitate regulated protein-unmixing of p53 and its regulators into PML nuclear condensates. In this review, we summarize and discuss the molecular mechanisms underlying PML nuclear condensate formation, and how these impact the biological function of p53 in driving the cell death and senescence responses. In addition, by using an in silico approach, we identify 299 proteins which share PML and p53 as binding partners, thus representing novel candidate proteins controlling p53 function and cell fate decision-making at the level of PML nuclear biocondensates. Full article

Human papillomaviruses (HPVs) inflict a significant burden on the human population. The clinical manifestations caused by high-risk HPV types are cancers at anogenital sites, including cervical cancer, as well as head and neck cancers. Host cell defense mechanisms such as autophagy are initiated upon HPV entry. At the same time, the virus modulates cellular antiviral processes and structures such as promyelocytic leukemia nuclear bodies (PML NBs) to enable infection. Here, we uncover the autophagy adaptor p62, also known as p62/sequestosome-1, as a novel proviral factor in infections by the high-risk HPV type 16 (HPV16). Proteomics, imaging and interaction studies of HPV16 pseudovirus-treated HeLa cells display that p62 is recruited to virus-filled endosomes, interacts with incoming capsids, and accompanies the virus to PML NBs, the sites of viral transcription and replication. Cellular depletion of p62 significantly decreased the delivery of HPV16 viral DNA to PML NBs and HPV16 infection rate. Moreover, the absence of p62 leads to an increase in the targeting of viral components to autophagic structures and enhanced degradation of the viral capsid protein L2. The proviral role of p62 and formation of virus-p62-PML hybrid bodies have also been observed in human primary keratinocytes, the HPV target cells. Together, these findings suggest the previously unrecognized virus-induced formation of p62-PML hybrid bodies as a viral mechanism to subvert the cellular antiviral defense, thus enabling viral gene expression. Full article

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4–6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML^−/−), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid–liquid phase separation (LLPS) leading to the formation of PML bodies. Full article

During initial infection, human papillomaviruses (HPV) take an unusual trafficking pathway through their host cell. It begins with a long period on the cell surface, during which the capsid is primed and a virus entry platform is formed. A specific type of clathrin-independent endocytosis and subsequent retrograde trafficking to the trans-Golgi network follow this. Cellular reorganization processes, which take place during mitosis, enable further virus transport and the establishment of infection while evading intrinsic cellular immune defenses. First, the fragmentation of the Golgi allows the release of membrane-encased virions, which are partially protected from cytoplasmic restriction factors. Second, the nuclear envelope breakdown opens the gate for these virus–vesicles to the cell nucleus. Third, the dis- and re-assembly of the PML nuclear bodies leads to the formation of modified virus-associated PML subnuclear structures, enabling viral transcription and replication. While remnants of the major capsid protein L1 and the viral DNA remain in a transport vesicle, the viral capsid protein L2 plays a crucial role during virus entry, as it adopts a membrane-spanning conformation for interaction with various cellular proteins to establish a successful infection. In this review, we follow the oncogenic HPV type 16 during its long journey into the nucleus, and contrast pro- and antiviral processes. Full article