The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML.

Acute promyelocytic leukaemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and the retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex (termed ND10, Kr bodies, nuclear bodies, PML oncogenic domains or PODs) which is disrupted in the APL disease state. PML contains a number of characterized motifs including a Zn2+ binding domain called the RING or C3HC4 finger. Here we describe the solution structure of the PML RING finger as solved by 1H NMR methods at physiological pH with r.m.s. deviations for backbone atoms of 0.88 and 1.39 A for all atoms. Additional biophysical studies including CD and optical spectroscopy, show that the PML RING finger requires Zn2+ for autonomous folding and that cysteines are used in metal ligation. A comparison of the structure with the previously solved equine herpes virus IE110 RING finger, shows significant differences suggesting that the RING motif is structurally diverse. The role of the RING domain in PML nuclear body formation was tested in vivo, by using site-directed mutagenesis and immunofluorescence on transiently transfected NIH 3T3 cells. Independently mutating two pairs of cysteines in each of the Zn2+ binding sites prevents PML nuclear body formation, suggesting that a fully folded RING domain is necessary for this process. These results suggest that the PML RING domain is probably involved in protein-protein interactions, a feature which may be common to other RING finger domains.     

PML isoform II plays a critical role in nuclear lipid droplet formation

Lipid droplets (LDs) in the nucleus of hepatocyte-derived cell lines were found to be associated with premyelocytic leukemia (PML) nuclear bodies (NBs) and type I nucleoplasmic reticulum (NR) or the extension of the inner nuclear membrane. Knockdown of PML isoform II (PML-II) caused a significant decrease in both nuclear LDs and type I NR, whereas overexpression of PML-II increased both. Notably, these effects were evident only in limited types of cells, in which a moderate number of nuclear LDs exist intrinsically, and PML-II was targeted not only at PML NBs, but also at the nuclear envelope, excluding lamins and SUN proteins. Knockdown of SUN proteins induced a significant increase in the type I NR and nuclear LDs, but these effects were cancelled by simultaneous knockdown of PML-II. Nuclear LDs harbored diacylglycerol O-acyltransferase 2 and CTP:phosphocholine cytidylyltransferase α and incorporated newly synthesized lipid esters. These results corroborated that PML-II plays a critical role in generating nuclear LDs in specific cell types.     

Herpesvirus protein ICP27 switches PML isoform by altering mRNA splicing

Viruses use alternative splicing to produce a broad series of proteins from small genomes by utilizing the cellular splicing machinery. Since viruses use cellular RNA binding proteins for viral RNA processing, it is presumable that the splicing of cellular pre-mRNAs is affected by viral infection. Here, we showed that herpes simplex virus type 2 (HSV-2) modifies the expression of promyelocytic leukemia (PML) isoforms by altering pre-mRNA splicing. Using a newly developed virus-sensitive splicing reporter, we identified the viral protein ICP27 as an alternative splicing regulator of PML isoforms. ICP27 was found to bind preferentially to PML pre-mRNA and directly inhibit the removal of PML intron 7a in vitro. Moreover, we demonstrated that ICP27 functions as a splicing silencer at the 3′ splice site of the PML intron 7a. The switching of PML isoform from PML-II to PML-V as induced by ICP27 affected HSV-2 replication, suggesting that the viral protein modulates the splicing code of cellular pre-mRNA(s) governing virus propagation.     

Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1

SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO–SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.     

PML control of cytokine signaling

The promyelocytic leukemia (PML) protein is a tumor suppressor acting as the organizer of nuclear matrix-associated structures named nuclear bodies (NBs). The involvement of PML in various cell processes, including cell death, senescence or antiviral defense underlines the multiple functions of PML due to its ability to interact with various partners either in the cytoplasm or in the nucleus. The importance of paracrine signaling in the regulation of PML expression is well established. More recently, a growing body of evidence also supports PML as a key regulator of cytokine signaling. These findings shed light on unsuspected biological functions of PML such as immune response, inflammation and cytokine-induced apoptosis. Here we review the current understanding of the pleiotropic activities of PML on cytokine-induced signaling.     

Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.     

The cell biology of disease: Acute promyelocytic leukemia, arsenic, and PML bodies

Acute promyelocytic leukemia (APL) is driven by a chromosomal translocation whose product, the PML/retinoic acid (RA) receptor α (RARA) fusion protein, affects both nuclear receptor signaling and PML body assembly. Dissection of APL pathogenesis has led to the rediscovery of PML bodies and revealed their role in cell senescence, disease pathogenesis, and responsiveness to treatment. APL is remarkable because of the fortuitous identification of two clinically effective therapies, RA and arsenic, both of which degrade PML/RARA oncoprotein and, together, cure APL. Analysis of arsenic-induced PML or PML/RARA degradation has implicated oxidative stress in the biogenesis of nuclear bodies and SUMO in their degradation.     

LAP2 binds to BAF.DNA complexes: requirement for the LEM domain and modulation by variable regions

LAP2 belongs to a family of nuclear membrane proteins sharing a 43 residue LEM domain. All LAP2 isoforms have the same N-terminal 'constant' region (LAP2-c), which includes the LEM domain, plus a C-terminal 'variable' region. LAP2-c polypeptide inhibits nuclear assembly in Xenopus extracts, and binds in vitro to barrier-to-autointegration factor (BAF), a DNA-bridging protein. We tested 17 Xenopus LAP2-c mutants for nuclear assembly inhibition, and binding to BAF and BAF small middle dotDNA complexes. LEM domain mutations disrupted all activities tested. Some mutations outside the LEM domain had no effect on binding to BAF, but disrupted activity in Xenopus extracts, suggesting that LAP2-c has an additional unknown function required to inhibit nuclear assembly. Mutagenesis results suggest that BAF changes conformation when complexed with DNA. The binding affinity of LAP2 was higher for BAF small middle dotDNA complexes than for BAF, suggesting that these interactions are physiologically relevant. Nucleoplasmic domains of XENOPUS: LAP2 isoforms varied 9-fold in their affinities for BAF, but all isoforms supershifted BAF small middle dotDNA complexes. We propose that the LEM domain is a core BAF-binding domain that can be modulated by the variable regions of LAP2 isoforms.     

An Arenavirus RING (Zinc-Binding) Protein Binds the Oncoprotein Promyelocyte Leukemia Protein (PML) and Relocates PML Nuclear Bodies to the Cytoplasm

The promyelocytic leukemia protein (PML) forms nuclear bodies which are altered in some disease conditions. We report that the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV) influences the distribution of PML bodies. In cells infected with LCMV, the Z protein and PML form large bodies primarily in the cytoplasm. Transient transfection studies indicate that Z alone is sufficient to redistribute PML to the cytoplasm and that PML and Z colocalize. Coimmunoprecipitation studies show specific interaction between PML and Z proteins. A similar result was observed with a Z protein from another arenavirus, Lassa virus, suggesting that this is a general feature of the Arenaviridae. Genetically engineered mutations in PML were used to show that the Z protein binds the N-terminal region of PML and does not need the PML RING or the nuclear localization signal to colocalize. The Z protein acts dominantly to overcome the diffuse phenotype observed in several PML mutants. The interaction between PML and Z may influence certain unique characteristics of arenavirus infection.