PML Nuclear Bodies Research Articles

HPV16 Entry into Epithelial Cells: Running a Gauntlet.

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.

3C protease of enterovirus 71 cleaves promyelocytic leukemia protein and impairs PML-NBs production

BackgroundEnterovirus 71 (EV71) usually infects infants causing hand-foot-mouth disease (HFMD), even fatal neurological disease like aseptic meningitis. Effective drug for preventing and treating EV71 infection is unavailable currently. EV71 3C mediated the cleavage of many proteins and played an important role in viral inhibiting host innate immunity. Promyelocytic leukemia (PML) protein, the primary organizer of PML nuclear bodies (PML-NBs), can be induced by interferon and is involved in antiviral activity. PML inhibits EV71 replication, and EV71 infection reduces PML expression, but the molecular mechanism is unclear.MethodsThe cleavage of PMLIII and IV was confirmed by co-transfection of EV71 3C protease and PML. The detailed cleavage sites were evaluated further by constructing the Q to A mutant of PML. PML knockout cells were infected with EV71 to identify the effect of cleavage on EV71 replication. Immunofluorescence analysis to examine the interference of EV71 3C on the formation of PML-NBs.ResultsEV71 3C directly cleaved PMLIII and IV. Furthermore, 3C cleaved PMLIV at the sites of Q430–A431 and Q444–S445 through its protease activity. Overexpression of PMLIV Q430A/Q444A variant exhibited stronger antiviral potential than the wild type. PMLIV Q430A/Q444A formed normal nuclear bodies that were not affected by 3C, suggesting that 3C may impair PML-NBs production via PMLIV cleavage and counter its antiviral activities. PML, especially PMLIV, which sequesters viral proteins in PML-NBs and inhibits viral production, is a novel target of EV71 3C cleavage.ConclusionsEV71 3C cleaves PMLIV at Q430–A431 and Q444–S445. Cleavage reduces the antiviral function of PML and decomposes the formation of PML-NBs, which is conducive to virus replication.

In This Issue

In This Issue

Molecular Cloning of Alternative Splicing Variants of the Porcine PML Gene and Its Expression Patterns During Japanese Encephalitis Virus Infection.

Promyelocytic leukemia (PML) protein is a crucial component of PML-nuclear bodies (PML-NBs). PML and PML-NBs are involved in the regulation of various cellular functions, including the antiviral immune response. The human PML gene can generate several different isoforms through alternative splicing. However, little is known about the porcine PML alternative splicing isoforms and their expression profiles during Japanese encephalitis virus (JEV) infection. In the present study, we cloned seven mature transcripts of porcine PML, all of which contained the same N-terminal sequence but differed in the C-terminal sequences due to alternative splicing. These seven transcripts encoded five proteins all of which had the RBCC motif and sumoylation sites. Amino acid sequence homology analysis showed that porcine PML-1 had relatively high levels of identity with human, cattle, and goat homologs (76.21, 77.17, and 77.05%, respectively), and low identity with the mouse homolog (61.78%). Immunofluorescence analysis showed that the typical PML-NBs could be observed after overexpression of the five PML isoforms in PK15 cells. Quantitative reverse transcription PCR (RT-qPCR) analysis showed significant upregulation of PML isoforms and PML-NB-associated genes (Daxx and SP100) at 36 and 48 h post-infection (hpi). Western blotting analysis indicated that the PML isoforms were upregulated during the late stage of infection. Moreover, the number of PML-NBs was increased after JEV infection. These results suggest that porcine PML isoforms may play essential roles in JEV infection.

Arsenic Trioxide Reprograms the Bone-Marrow Microenvironment to Sensitize Minimal Residual Disease in Acute Myeloid Leukemia

Eliminating minimal residual disease (MRD) in order to prevent relapse is an unmet need in acute myeloid leukemia (AML). One of the success stories is acute promyelocytic leukemia (APL), a unique subtype of AML. The use of arsenic trioxide (ATO) in combination with all-trans retinoic acid (ATRA) has essentially eliminated disease relapse (<1% ATRA+ATO vs. ~18% ATRA+ chemotherapy - NEJM (2013), 369(2), 111-121). This is even more impressive as neither agent alone results in significant cure of patients with APL. In fact, single agent ATRA induces remission without cure due to bone marrow stroma expression of CYP26 enzymes (PloS one (2015), 10(6), e0127790), which are induced by ATRA, inactivate retinoids and protect APL MRD (Leukemia (2020), 34(11), 3077-3081). While ATO may synergize with ATRA by restoring PML nuclear bodies (Nature medicine (2014), 20(2), 167-174) and rescuing RAR-α dependent transcription (Nature reviews. Cancer (2018), 18(2), 117-127) in the bulk of the APL tumor cells, it is unclear how these mechanisms contribute to elimination of MRD, particularly given the hypothesized lack of ATRA in the bone marrow niche. It was previously reported that combination of ATRA with ATO may be associated with high incidence of transaminitis and headaches (NEJM (2013), 369(2), 111-121). Since these may be clinical signs of ATRA toxicity, we investigated if addition of ATO changes the pharmacokinetics (PKs) of ATRA in patients with APL. We determined the area under the plasma concentration versus time curve (AUC) for ATRA in patients suspected of APL and treated with single agent ATRA (“preATO”) and in the same patients after the diagnoses of APL was confirmed and the patients received ATO (“postATO”). Even though ATRA should have an expected drop in AUC during the first 3-4 days of therapy (JPET (2017), 361(2), 246-258), addition of ATO resulted in improved ATRA PKs (Figure A). Since ATRA levels are mainly controlled by clearance via hepatic CYP26s, we tested if treatment with ATO changes the levels of CYP26 or its induction by ATRA. We found that exposure to ATO decreases both baseline and ATRA-induced upregulation of CYP26B1 in HepG2 cells (Figure B). Similarly, ATO decreases CYP26B1 levels in human bone marrow derived mesenchymal stroma cells (MSCs) at baseline (21%±7% of NO ATO control, n=3, p<0.01) and after ATRA-induced upregulation (66%±14% of ATRA only control, n=3, p<0.01). These changes resulted in improved ATRA PKs in vitro in the presence of bone marrow MSCs (Figure C). Next, we used NB4-EvAsR cells (NB4R), ATO-resistant APL cells to test if ATO-induced improvement of MSC-dependent ATRA PKs may contribute to elimination of APL MRD. We found that ATO sensitizes NB4R to ATRA in the presence of BM MSCs but not in stroma free conditions (Figure D). ATO had no effect on ATRA-induced differentiation of NB4R in the presence of CYP26B1 knockout stroma (this stroma offers no protection from ATRA) or if stroma expresses CYP26B1 under the control of a viral promoter and thus, resistant to ATO downregulation. This data suggest that ATO-induced changes of BM MSC expression of CYP26 may contribute to elimination of MRD in APL. Stromal CYP26 protects not only APL but also non-APL AML cells from retinoids (Leukemia (2020), 34(11), 3077-3081). Retinoids are synergistic with FLT3 inhibitors to eliminate mutant FLT3 AML cells (Blood (2016), 127(23), 2867-2878) and stromal CYP26 breaks this synergism (Blood (2015) 126 (23): 790). We tested if ATO sensitizes mutant FLT3 AML cells, MV4;11 to gilteritinib in the presence of BM MSCs. For this, we derived MV4.11 cells resistant to ATO (MV4.11R) via long term exposure to increasing concentration of ATO. As seen with NB4R, ATO treatment synergizes with gilteritinib to decrease the IC50 of gilteritinib against the clonogenic activity of MV4.11R in the presence of stroma but not in stroma free conditions (Figure E). In an effort to fully explore the translational potential of these findings, we are currently investigating the mechanism by which ATO changes CYP26 levels.Taken together these findings support a model in which ATO changes the bone marrow niche and it's ability to metabolize retinoids and thus, sensitizes MRD in AML to targeted therapy. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Ghiaur: Syros Pharmaceuticals: Consultancy; Menarini Richerche: Research Funding.

Implication of the nuclear trafficking of rabies virus P3 protein in viral pathogenicity.

Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.

Chromatin mobility and relocation in DNA repair.

The nucleus is a dynamic environment containing chromatin, membraneless organelles, and specialized molecular structures at the nuclear membrane. Within the spectrum of DNA repair activities are observations of increased mobility of damaged chromatin and the displacement of DNA lesions to specific nuclear environments. Here, we focus on the role that nuclear-specific filamentous actin plays in mobilizing damaged chromatin in response to DNA double-strand breaks and replication stress. We also examine nuclear pore complexes and promyelocytic leukemia-nuclear bodies as specialized platforms for homology-directed repair. The literature suggests an emerging model where specific types of DNA lesions are subjected to nuclear-derived forces that mobilize damaged chromatin and promote interaction with repair hubs to facilitate specialized repair reactions.

A Tale of Usurpation and Subversion: SUMO-Dependent Integrity of Promyelocytic Leukemia Nuclear Bodies at the Crossroad of Infection and Immunity.

Promyelocytic leukemia nuclear bodies (PML NBs) are multi-protein assemblies representing distinct sub-nuclear structures. As phase-separated molecular condensates, PML NBs exhibit liquid droplet-like consistency. A key organizer of the assembly and dynamics of PML NBs is the ubiquitin-like SUMO modification system. SUMO is covalently attached to PML and other core components of PML NBs thereby exhibiting a glue-like function by providing multivalent interactions with proteins containing SUMO interacting motifs (SIMs). PML NBs serve as the catalytic center for nuclear SUMOylation and SUMO-SIM interactions are essential for protein assembly within these structures. Importantly, however, formation of SUMO chains on PML and other PML NB-associated proteins triggers ubiquitylation and proteasomal degradation which coincide with disruption of these nuclear condensates. To date, a plethora of nuclear activities such as transcriptional and post-transcriptional regulation of gene expression, apoptosis, senescence, cell cycle control, DNA damage response, and DNA replication have been associated with PML NBs. Not surprisingly, therefore, SUMO-dependent PML NB integrity has been implicated in regulating many physiological processes including tumor suppression, metabolism, drug-resistance, development, cellular stemness, and anti-pathogen immune response. The interplay between PML NBs and viral infection is multifaceted. As a part of the cellular antiviral defense strategy, PML NB components are crucial restriction factors for many viruses and a mutual positive correlation has been found to exist between PML NBs and the interferon response. Viruses, in turn, have developed counterstrategies for disarming PML NB associated immune defense measures. On the other end of the spectrum, certain viruses are known to usurp specific PML NB components for successful replication and disruption of these sub-nuclear foci has recently been linked to the stimulation rather than curtailment of antiviral gene repertoire. Importantly, the ability of invading virions to manipulate the host SUMO modification machinery is essential for this interplay between PML NB integrity and viruses. Moreover, compelling evidence is emerging in favor of bacterial pathogens to negotiate with the SUMO system thereby modulating PML NB-directed intrinsic and innate immunity. In the current context, we will present an updated account of the dynamic intricacies between cellular PML NBs as the nuclear SUMO modification hotspots and immune regulatory mechanisms in response to viral and bacterial pathogens.

Modulation of H3.3 chromatin assembly by PML: A way to regulate epigenetic inheritance.

Although the promyelocytic leukemia (PML) protein is renowned for regulating a wide range of cellular processes and as an essential component of PML nuclear bodies (PML-NBs), the mechanisms through which it exerts its broad physiological impact are far from fully elucidated. Here, we review recent studies supporting an emerging view that PML's pleiotropic effects derive, at least partially, from its role in regulating histone H3.3 chromatin assembly, a critical epigenetic mechanism. These studies suggest that PML maintains heterochromatin organization by restraining H3.3 incorporation. Examination of PML's contribution to H3.3 chromatin assembly in the context of the cell cycle and PML-NB assembly suggests that PML represses heterochromatic H3.3 deposition during S phase and that transcription and SUMOylation regulate PML's recruitment to heterochromatin. Elucidating PML' s contributions to H3.3-mediated epigenetic regulation will provide insight into PML's expansive influence on cellular physiology and open new avenues for studying oncogenesis linked to PML malfunction.

Structure, Activity and Function of the SETDB1 Protein Methyltransferase.

The SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1) is a prominent member of the Suppressor of Variegation 3–9 (SUV39)-related protein lysine methyltransferases (PKMTs), comprising three isoforms that differ in length and domain composition. SETDB1 is widely expressed in human tissues, methylating Histone 3 lysine 9 (H3K9) residues, promoting chromatin compaction and exerting negative regulation on gene expression. SETDB1 has a central role in normal physiology and nervous system development, having been implicated in the regulation of cell cycle progression, inactivation of the X chromosome, immune cells function, expression of retroelements and formation of promyelocytic leukemia (PML) nuclear bodies (NB). SETDB1 has been frequently deregulated in carcinogenesis, being implicated in the pathogenesis of gliomas, melanomas, as well as in lung, breast, gastrointestinal and ovarian tumors, where it mainly exerts an oncogenic role. Aberrant activity of SETDB1 has also been implicated in several neuropsychiatric, cardiovascular and gastrointestinal diseases, including schizophrenia, Huntington’s disease, congenital heart defects and inflammatory bowel disease. Herein, we provide an update on the unique structural and biochemical features of SETDB1 that contribute to its regulation, as well as its molecular and cellular impact in normal physiology and disease with potential therapeutic options.

Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers.

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs). IE1 interacts directly with PML, the key protein of PML-NBs, through its core domain and disrupts the dot-like multiprotein complexes thereby abrogating the antiviral effects. Here we present the crystal structures of the human and rat cytomegalovirus core domain (IE1CORE). We found that IE1CORE domains, also including the previously characterized IE1CORE of rhesus CMV, form a distinct class of proteins that are characterized by a highly similar and unique tertiary fold and quaternary assembly. This contrasts to a marked amino acid sequence diversity suggesting that strong positive selection evolved a conserved fold, while immune selection pressure may have fostered sequence divergence of IE1. At the same time, we detected specific differences in the helix arrangements of primate versus rodent IE1CORE structures. Functional characterization revealed a conserved mechanism of PML-NB disruption, however, primate and rodent IE1 proteins were only effective in cells of the natural host species but not during cross-species infection. Remarkably, we observed that expression of HCMV IE1 allows rat cytomegalovirus replication in human cells. We conclude that cytomegaloviruses have evolved a distinct protein tertiary structure of IE1 to effectively bind and inactivate an important cellular restriction factor. Furthermore, our data show that the IE1 fold has been adapted to maximize the efficacy of PML targeting in a species-specific manner and support the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV.

TRAIL-receptor 2\u2014a novel negative regulator of p53

TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2) can induce apoptosis in cancer cells upon crosslinking by TRAIL. However, TRAIL-R2 is highly expressed by many cancers suggesting pro-tumor functions. Indeed, TRAIL/TRAIL-R2 also activate pro-inflammatory pathways enhancing tumor cell invasion, migration, and proliferation. In addition, nuclear TRAIL-R2 (nTRAIL-R2) promotes malignancy by inhibiting miRNA let-7-maturation. Here, we show that TRAIL-R2 interacts with the tumor suppressor protein p53 in the nucleus, assigning a novel pro-tumor function to TRAIL-R2. Knockdown of TRAIL-R2 in p53 wild-type cells increases the half-life of p53 and the expression of its target genes, whereas its re-expression decreases p53 protein levels. Interestingly, TRAIL-R2 also interacts with promyelocytic leukemia protein (PML), a major regulator of p53 stability. PML-nuclear bodies are also the main sites of TRAIL-R2/p53 co-localization. Notably, knockdown or destruction of PML abolishes the TRAIL-R2-mediated regulation of p53 levels. In summary, our finding that nTRAIL-R2 facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors.

Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression.

Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.

SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data.

The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1–3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.

PML-NB-dependent type I interferon memory results in a restricted form of HSV latency.

Herpes simplex virus (HSV) establishes latent infection in long-lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency are unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also find that the subnuclear condensates, promyelocytic leukemia nuclear bodies (PML-NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV-1 genomes colocalize with PML-NBs throughout a latent infection of neurons only when type I IFN is present during initial infection. Depletion of PML prior to or following infection does not impact the establishment latency; however, it does rescue the ability of HSV to reactivate from IFN-treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate.

SUMO: Glue or Solvent for Phase-Separated Ribonucleoprotein Complexes and Molecular Condensates?

Spatial organization of cellular processes in membranous or membrane-less organelles (MLOs, alias molecular condensates) is a key concept for compartmentalizing biochemical pathways. Prime examples of MLOs are the nucleolus, PML nuclear bodies, nuclear splicing speckles or cytosolic stress granules. They all represent distinct sub-cellular structures typically enriched in intrinsically disordered proteins and/or RNA and are formed in a process driven by liquid-liquid phase separation. Several MLOs are critically involved in proteostasis and their formation, disassembly and composition are highly sensitive to proteotoxic insults. Changes in the dynamics of MLOs are a major driver of cell dysfunction and disease. There is growing evidence that post-translational modifications are critically involved in controlling the dynamics and composition of MLOs and recent evidence supports an important role of the ubiquitin-like SUMO system in regulating both the assembly and disassembly of these structures. Here we will review our current understanding of SUMO function in MLO dynamics under both normal and pathological conditions.

The viral SUMO‐targeted Ubiquitin Ligase ICP0 is phosphorylated and activated by host kinase Chk2

When the Herpes Simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called PML Nuclear Bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a SUMO-targeted Ubiquitin ligase (STUbL) ICP0 that degrades PML NBs to alleviate the repression. The molecular details of the mechanism used by ICP0 to target PML NBs are unclear. Here we identify a bonafide SUMO-Interacting motif in ICP0 (SLS4) that is essential and sufficient to target SUMOylated proteins in PML NBs like the PML and Sp100. Phosphorylation of SLS4 creates new salt-bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity, and switches ICP0 into a potent STUbL. HSV activates ATM/Chk2 pathway to regulate the cell-cycle of the host. We report that the activated Chk2 also phosphorylates ICP0 at SLS4 and enhances its STUbL activity. Our results uncover that a viral STUbL counters antiviral response by exploiting an unprecedented cross-talk of three post-translational modifications; ubiquitination, SUMOylation, and phosphorylation.

Amyloid precursor protein elevates fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates consisting of its adapter protein FE65, the histone acetyltransferase TIP60 and the tumour suppressor proteins p53 and PML. APP C-terminal (APP-CT50) complexes co-localize and co-precipitate with p53 and PML. The PML nuclear body generation is induced and fusion occurs over time depending on APP signalling and STED imaging revealed active gene expression within the complex. We further show that the nuclear aggregates of APP-CT50 fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimer’s disease brains reveal a highly significant reduction of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT50 signalling to the nucleus takes place in the aged human brain and is involved in the pathophysiology of AD.

Chemical Dimerization-Induced Protein Condensates on Telomeres.

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

Promyelocytic leukemia protein promotes the phenotypic switch of smooth muscle cells in atherosclerotic plaques of human coronary arteries.

Promyelocytic leukemia protein (PML) is a constitutive component of PML nuclear bodies (PML-NBs), which function as stress-regulated SUMOylation factories. Since PML can also act as a regulator of the inflammatory and fibroproliferative responses characteristic of atherosclerosis, we investigated whether PML is implicated in this disease. Immunoblotting, ELISA and immunohistochemistry showed a stronger expression of PML in segments of human atherosclerotic coronary arteries and sections compared with non-atherosclerotic ones. In particular, PML was concentrated in PML-NBs from α-smooth muscle actin (α-SMA)-immunoreactive cells in plaque areas. To identify possible functional consequences of PML-accumulation in this cell type, differentiated human coronary artery smooth muscle cells (dHCASMCs) were transfected with a vector containing the intact PML-gene. These PML-transfected dHCASMCs showed higher levels of small ubiquitin-like modifier (SUMO)-1-dependent SUMOylated proteins, but lower levels of markers for smooth muscle cell (SMC) differentiation and revealed more proliferation and migration activities than dHCASMCs transfected with the vector lacking a specific gene insert or with the vector containing a mutated PML-gene coding for a PML-form without SUMOylation activity. When dHCASMCs were incubated with different cytokines, higher PML-levels were observed only after interferon γ (IFN-γ) stimulation, while the expression of differentiation markers was lower. However, these phenotypic changes were not observed in dHCASMCs treated with small interfering RNA (siRNA) suppressing PML-expression prior to IFN-γ stimulation. Taken together, our results imply that PML is a previously unknown functional factor in the molecular cascades associated with the pathogenesis of atherosclerosis and is positioned in vascular SMCs (VSMCs) between upstream IFN-γ activation and downstream SUMOylation.