PML body
PML bodies (PML body, PML body, PML oncogenic domain (POD), nuclear domain 10 (ND 10) or Kremer (Kr) body) are spherical structures with a diameter of 0.1–1.0 µm that occur in the nucleus in most cell lines and many tissues. PML bodies have the ability to recruit a wide variety of seemingly independent proteins. In the nuclei of vertebrate cell culture cells, an average of 5 to 30 PML nuclear bodies with a size of ~ 0.2 to 1 µm are found.
The name PML body is derived from acute promyelocyte leukemia , as they are destroyed in this disease, but also in various viral infections. PML bodies were discovered in the early 1960s. The identification of PML, a gene involved in oncogenic chromosomal translocation , as the key organizer of these objects aroused further interest in them. The various levels of regulation by a specific post-translational modification , SUMOylation, have raised several unsolved problems. Functionally, PML bodies can bind, modify or break down partner proteins.
Several sub-types of these bodies have been defined on a morphological basis. They all have an electron-tight shell and contain an inner core. PML bodies came to the fore with the observation that the oncogenic PML / RARA protein destroys them in a treatment-reversible manner. PML bodies are regulated by cellular stress: viral infection, DNA damage, transformation, and oxidative stress . In addition, the transcription of PML and several genes that code for partner proteins is dramatically improved by interferons .
history
The first research in this field was carried out with the help of electron microscopy and the use of autoantibodies. In the early 1960s, researchers demonstrated the presence of dense spherical objects using electron microscopy. Two classes of bodies were then described: void (fibrillar) and granular bodies. The granular bodies contain a microgranular material inside, for which ribonucleoproteins have been suggested. PML nuclear bodies were later observed by the immunofluorescence method with autoimmune sera from patients with primary biliary cirrhosis . These enabled the identification of the first associated protein, SP100, and an initial characterization of these structures.
The identification of an anti-nuclear matrix antibody that labeled the same structures as SP100 established the first link between these bodies and the nuclear matrix. The localization of PML proteins to the same sites as SP100 led to renewed interest in these areas. PML is a protein that is fused with the retinoic acid receptor α (RARA) in the PML / RARA oncoprotein of acute promyelocytic leukemia (APL).
In the 1990s it was observed that PML / RARA interfered with these areas in APL cells. This led to more intensive research on these bodies in the scientific community. In addition, PML bodies were restored through two different anti-APL therapies, retinoic acid and arsenic trioxide . This triggered the PML / RARA dismantling later. This made it possible to identify the first noticeable parallel between the status of the body and that of the cell. Many other studies in the 2000s showed that PML bodies changed under stressful conditions, particularly viral infections, heat shock, and exposure to heavy metals.
Proteins involved
PML bodies are recruiting an ever-increasing number of partner proteins (now in the region of 100), with one of the best-studied being DAXX, a powerful repressor of transcription and modulator of apoptosis . It is crucial that PML is the real organizer of this body. Among these recruited proteins, a special mention deserves: a ubiquitin-like protein called SUMO , as PML conjugation through SUMO plays a critical role in recruiting partners, many of whom are sumoylated themselves.
PML
B : B box, CC : coiled-coil domain, NLS : nuclear localization signal, S : sumoylation sites, SIM : SUMO interaction motif. Bottom: Representation of the six nuclear PML isoforms (I to VI). Exons 1-6 are shared by all isoforms, while their C-termini differ individually due to the alternative splicing of exons 7-9
The PML protein is a tumor suppressor protein from the TRIM / RBCC protein family and the organizer of the PML core bodies. Like many proteins in this family, the PML protein is a ubiquitin ligase . This can create subcellular structures through auto-assembly. The transcription of the PML gene is controlled by interferons α / β or γ, but also by the p53 protein. Both cause a dramatic increase in the number and size of PML bodies.
PML has an amino-terminal RING finger domain that binds a SUMO ligase directly, and a coiled-coil (double helix structure) that mediates homodimerization . This section of the protein is called the RBCC ( RING finger, B box and coiled coil domain ). It also has a protein segment called SIM (SUMO interaction motif).
By alternative splicing of several variants (isoforms) of the PML protein are prepared. These have different carboxy terminal domains. In addition to the nuclear localization signal (NLS), which is present in all PML isoforms, PML-I has a nuclear export signal that enables all isoforms to be moved back and forth between the nucleus and cytoplasm through heterodimer formation. The most common isoform, PML-I, also harbors an exonuclease III domain. All six isoforms are expressed in human cells. The PML isoforms I and II make up the largest share.
PML undergoes several critical post-translational modifications, most notably through phosphorylation and sumoylation. PML sumoylation is particularly important for building PML bodies. Various kinases , activated by damage to the DNA or by stress, phosphorylate PML. In doing so, they may regulate PML stability, the biogenesis of nuclear bodies and contribute to DNA repair or apoptosis control.
The vast majority of the PML protein pool in a cell is not bound in the PML bodies. In most cell lines, more than 90% of PML proteins can be found distributed in the nucleus.
| PML isoform | Localization in pml - / - cells |
|---|---|
| PML I | Nuclear in stress, cytoplasm |
| PML II | Fibrillar / nuclear envelope (lamina?) |
| PML IV | Nuclear in stress |
| PMLV | Thick shell / anchorage in the matrix |
| PMLVII | Cytoplasmic / early endosomes |
The expression of PML isoforms in pml - / - cells reveals their own localizations. This suggests that the carboxy terminus controls interactions with specific but still unknown partners.
SUMO
SUMO proteins are a family of small proteins that are covalently linked to other proteins in cells to change their function. Sumoylation is a post-translational modification that is involved in various cellular processes, such as: B. chromatin organization , transcription , signal transduction , apoptosis , protein stability and response to stress.
SP100
This gene codes for a subnuclear organelle and major component of the PML body. PML and SP100 are covalently modified by the SUMO-1 modifier. The encoded protein binds heterochromatin proteins and is believed to play a role in tumorigenesis, immunity and gene regulation. Alternatively, spliced variants of this gene have been identified, one of which encodes a highly mobile group protein.
DAXX
DAXX ( Death-associated protein 6 ) is a protein associated with the death domain . It was first discovered through its cytoplasmic interaction with the Fas receptor , which initiates cell apoptosis. It has been associated with heterochromatin and PML bodies and is involved in many core processes, including transcription and cell cycle regulation.
p53
As a transcription factor after DNA damage, the human tumor suppressor p53 regulates the expression of genes that are involved in the control of the cell cycle, in the induction of apoptosis (programmed cell death) or in DNA repair.
structure
Types of PML bodies
It is believed that there are several types of PML bodies. In fact, the PML protein aggregates in different forms in response to a variety of stresses. The most extensively studied factor affecting PML distribution is arsenic trioxide. Several DNA damage activated kinases have also been recognized as important. Stress-induced aggregation can promote the aggregation of typical nuclear bodies or, conversely, break them down into micro-bodies. The differences between these types of PML bodies are based on shape or content. This leads to a more dynamic view of the PML bodies than previously thought.
| Stress factor | PML NB appearance | Cell types |
|---|---|---|
| IFNs | Increased number and size | All |
| As 2 O 3 | Large PML envelope, reduced number | All |
| CdCl 2 / heat shock | Distributed micro-bodies | All |
| Proteasome inhibitor | Increase in the nucleolus | Primary cell lines |
| Increased number and size | Transformed cell lines | |
| Actinomycin D. | Large, near the nucleolus | Primary and transformed cell lines |
| Distributed micro-bodies | Transformed cell lines | |
| Ionizing radiation (gamma, UVc) | Large, near the nucleolus | Primary and transformed cell lines |
Structure of a common PML body
The classic PML body is a spherical object with a diameter of 0.1–1 µm that may or may not have a granular center. These bodies, from five to 15 per nucleus, are mostly proteinaceous and generally contain no RNA or DNA. The PML protein forms the outer shell of these bodies and the partner proteins are usually located inside. This can easily be shown in the case of overexpression of PML or its partner proteins. Proteins can pass through this shell with only moderate restriction of their mobility.
Like several other bodies in the nucleus, PML nuclear bodies are also present in the interchromosomal space. This probably explains why they are often found around or around other bodies. Although free of DNA, PML bodies can be associated with some specific chromosomal loci, such as: B. the MHC class I gene cluster region. For these, the PML bodies for modulating chromatin architecture and transcription were proposed. An elegant study has shown that a PML body is constantly confronted with a repressor locus, which underscores the relationship with transcriptional regulation. Conversely, chromatin changes that occur during transcription or the cell cycle can change the structure and number of PML bodies. PML bodies are greatly altered during many viral infections. For example, they can accumulate viral genomes on their periphery or in their central core during infection of dormant cells.
In functionally specialized PML bodies, such as B. in the alternative extension of telomere- associated PML core bodies (APBs), the inner core of PML bodies contains chromatin, in this case telomere DNA.
In cells of immunodeficiency, centromeric instability, and facial dysmorphism (ICF) syndrome , the inner core of the PML body contains pericentric satellite heterochromatin of chromosome 1.
education
The formation and structural integrity of PML bodies is based on at least five basic principles:
1) an oxidation-driven disulfide crosslinking of PML
2) the self-oligomerizing properties of the PML-RBCC motif,
3) the poly-SUMO chains on the three main target lysines
4) the non-covalent interaction of SUMO with SUMO interacting motifs (SIM) in nuclear body-associated factors and
5) specific sequences in different PML protein isoforms.
In the first step of assembly, oxidized PML monomers enable the formation of disulfide-linked covalent multimers. These organize themselves in the outer shell of the core body. The non-covalent homodimerization mediated by the RBCC domain can be similarly important for the early PML core body assembly step.
Then poly-sumoylations, SUMO / SIM interactions and the addition of SUMO- and / or SIM-containing binding partners form a mature PML body with a peripheral framework, consisting of the six different PML isoforms, their SIM motifs and the poly -SUMO chains.
The PML core body structure offers a multitude of potential sites to which an assortment of PML-interacting, SIM-containing and / or sumoylated partner proteins can temporarily bind to a greater or lesser extent. The different residence times of the binding partners in the PML bodies depend on the number and strength of their individual interaction modules. This is consistent with the presence of multiple sumoylation sites and SIMs in key PML body components such as PML, SP100, DAXX, HIPK2, UBC9, PIASy, and RNF4.
dynamics
Studies have shown that PML is a stable part of the body and that partner proteins are more mobile, although they are temporarily held in the core bodies. The exchange rates of the different PML isoforms between nuclear body and caryoplasm showed a clear difference for the PML-V isoform. This forms peculiar thick-shelled core bodies and could anchor the bodies in the core matrix. The bodies themselves are not very mobile, although fusions and splits can be observed through the course of the cell cycle. Analyzes of the nuclear bodies during the cell cycle have provided evidence of duplication by a cleavage mechanism during the S phase . They also analyzed the new body formation during the M / G1 transition . During mitosis , the PML proteins remain aggregated but become phosphorylated, desumoylated, and release their partners. Before the nuclear membrane disintegrates in the prometaphase, PML bodies lose their chromatin connection, which leads to increased mobility. PML associates with nuclear membranes and nucleoporins during mitosis, which facilitates the regeneration of the nuclear envelope during the transition from telophase / G1. Finally, during the transition from telophase to G1, SP100 and DAXX re-enter the core and then bind to the preformed bodies, SP100 first and DAXX later.
Aside from arsenic trioxide, which promotes core body formation, heat shock or heavy metals induce reversible core body fragmentation through the formation of highly mobile micro-bodies that are free from SUMO and most partners. Single cell studies after coping with stress have shown that bodies were somehow restored to their initial size, location, and number, suggesting that PML bodies may assemble in predetermined locations.
The cell cycle-dependent dismantling of PML bodies begins with the de-sumoylation of PML at the beginning of mitosis. The spherical shell structure of the PML bodies collapses and other components such as SUMO, SP100 and DAXX loosen or are removed. During mitosis, PML aggregates into so-called mitotic collections of PML protein (MAPPs).
Functions
The functional diversity of the transient PML body components is likely the basis for the many different biological roles assigned to these core structures. PML bodies have been functionally linked to apoptosis, nuclear proteolysis, senescence , stem cell renewal, regulation of gene expression, tumor suppression, DNA damage response , telomere elongation and stability, epigenetic regulation, and antiviral responses. In addition, they play a role in controlling the cell cycle, cellular stress response, DNA repair, and protein modification processes. Generally speaking, the various aspects of PML body functions primarily indicate their role in maintaining the genome.
One hypothesis for integrating all of these functions into a unified concept is based on the idea that PML bodies provide a stable protein framework on which the binding partners associate for their efficient post-translational modification or sequestration. The controlled accumulation or release of specific nuclear factors from the bodies can improve their functional interaction on the basis of measures governed by mass laws and thereby refine the signal cascades through the karyoplasm.
A research report suggests that phase-separated liquid droplet structures can form in living cells through the PolySUMO / polySIM interface in PML bodies. PML bodies belong to the family of viscous, membrane-free core spaces that can act as phase separation condensates that correspond to the lipid droplets.
The biochemical environment within a phase-separating PML body is different from that in the surrounding caryoplasm, and this difference could enable unique strategies to regulate nuclear pathways, including regulation of enzyme reaction kinetics (i.e. post-translational modifications), regulation of the specificity of biochemical reactions, sequestration of Molecules and the buffering of the cell concentration of molecules.
DNA repair : The PML protein is phosphorylated by several kinases activated by DNA damage. Several studies have shown that PML bodies are involved in DNA repair by recruiting different proteins or by releasing them from core bodies. Some of these proteins are localized in nuclear bodies under non-stressed conditions, while others are only associated with the nuclear bodies after DNA damage. In response to DNA damage, PML bodies seem to capture the damaged areas and, through a cleavage mechanism, form an increased number of micro-bodies. The exact role of PML (viewer or actor) in these various processes is still unclear.
Post-translational modification of partner proteins : Perhaps the most studied post-translational modifications were those of the tumor suppressor p53. A striking finding was the concentration of p53-modifying enzymes (CBP, HDM2, HIPK2 and HAUSP) within the core body. PML-enhanced acetylation, sumoetylation, and phosphorylation in the core bodies all appear to improve p53 function.
The kinase activity of some proteins can be influenced by the translocation into the PML bodies: auto-phosphorylation and dephosphorylation of kinases have been observed. There is also some evidence that PML can directly improve protein sumoylation in yeast. The PML core bodies have been proposed to improve sumoylation of specific partner proteins. Since many core body-associated proteins contain a SIM, this can improve partner sequestration within the core body.
Partner sequestration : Sequestration or "emplacement" was the first proposed function of PML bodies. This sequestration is reflected in the relative accumulation of the nucleoplasmic and the nuclear body-associated form of PML partners, which varies greatly between the individual partners and the levels of PML expression and sumoylation. A well-studied, segregated partner is DAXX, a powerful repressor that divides between sumolylated proteins, including PML and many transcription factors. The sequestration of DAXX by nuclear body-associated and sumoylated PMLs releases the transcriptional repression by DNA-bound sumoylated transcription factors. The sequestration of DAXX also regulates apoptosis.
Partner protein breakdown : Several unstable proteins have been located on PML bodies, while proteins that are impaired in their breakdown accumulate with PML, SUMO and ubiquitin. The PML breakdown induced by arsenic trioxide has established a link between PML bodies and protein breakdown. Arsenic trioxide triggers an initial sumoylation of K160, followed by proteasome-dependent degradation.
Tumor suppressor : In addition to the correlative relationship between carcinogenesis and PML expression, there are many experimental indications of a direct tumor suppressive role of PML. Several independent studies have shown that overexpression of PML can slow or block cell cycle progression in a variety of cancer cell lines. Further analyzes of typical stress reaction pathways showed the involvement of the tumor suppressors pRb and p53 in the PML overexpression-induced cellular senescence. The molecular details of the PML effect along the pRB and / or p53 tumor suppressive pathways remain elusive. In addition to cellular senescence, PML plays an essential functional role in apoptosis. PML loss is correlated with the progression of many cancers, and in most cases, low PML expression is associated with a poor prognosis. The tumor suppressor function of PML bodies can be linked to their ability to accumulate many proteins that are involved in responding to DNA damage and repair pathways. This presumably stabilizes DNA repair complexes and enhances their activities. Although the physiological function of PML and the PML nucleus is not yet fully understood, their tumor suppressive role in assisting DNA damage progression may be common to all of these potential functions.
Regulation of transcription : There are various indications for the influence of PML and PML corpuscles on transcription regulation. A study of the molecular interactions in the cell revealed 166 different proteins that can interact with PML physically or functionally. Half of these proteins are directly or indirectly involved in the control of transcription. Transcription factors such as CBP / p300, Daxx and p53 temporarily localize to the PML corpuscles, where they are modified and / or compartmentalized. The PML isoform IV can also act as a transcriptional regulator when artificially recruited to promoters.
Antiviral Response: PML bodies are able to mediate intrinsic repression of viral replication by inducing epigenetic silencing of viral genomes or through entrapment mechanisms. In addition, PML bodies develop as co-activators of cellular genes that exert antiviral activities such as cytokines and ISGs. Components of the PML body are upregulated even by IFN treatment. To overcome these antiviral activities, many viruses have developed antagonistic proteins that affect individual PML body components or the entire cell structure. The proteins of human papilloma viruses can specifically bind to some PML isoforms, so that they can be subjected to proteasomal degradation. The adenovirus protein also specifically interacts with a PML isoform. This interaction leads to the proteasomal degradation of PML and thus to the dissolution of the nuclear bodies.
Alternative extension of telomeres : The unlimited growth potential of cancer cells requires the maintenance of their telomeres. This is often achieved by reactivating telomerase. However, an alternative extension of the telomere (ALT) mechanism is active in a significant proportion of the tumors. The molecular mechanism of the ALT pathway remains elusive. In particular, the role of the characteristic complexes of PML bodies with telomeres, the ALT-associated PML bodies (APBs), is currently being investigated. PML bodies in normal cells do not contain nucleic acids. In ALT-positive cells, however, a subset of PML bodies with telomere DNA localizes.
Individual evidence
- ↑ a b c d e f g h i j k V. Lallemand-Breitenbach, H. de The: PML Nuclear Bodies . In: Cold Spring Harbor Perspectives in Biology . tape 2 , no. 5 , May 1, 2010, ISSN 1943-0264 , p. a000661 – a000661 , doi : 10.1101 / cshperspect.a000661 , PMID 20452955 , PMC 2857171 (free full text) - ( cshlp.org [accessed March 13, 2019]).
- ↑ a b c Tobias Ulbricht: The role of PML corpuscles in the regulation of MHC class II expression. (PDF) In: Dissertation. March 31, 2010, accessed April 2, 2019 .
- ↑ a b c d e f g h Peter Hemmerich, Klaus Weisshart, Shamci Monajembashi, Christian Hoischen: Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies . In: Frontiers in Oncology . tape 8 , 2018, ISSN 2234-943X , doi : 10.3389 / fonc.2018.00125 ( frontiersin.org [accessed April 5, 2019]).
- ↑ sp100 protein, SP100 nuclear antigen - Creative BioMart. Retrieved April 7, 2019 .
- ↑ M. Lang, T. Jegou, I. Chung, K. Richter, S. Munch: Three-dimensional organization of promyelocytic leukemia nuclear bodies . In: Journal of Cell Science . tape 123 , no. 3 , February 1, 2010, ISSN 0021-9533 , p. 392-400 , doi : 10.1242 / jcs.053496 ( biologists.org [accessed April 13, 2019]).
- ↑ Toshiro Tsukamoto, Noriyo Hashiguchi, Susan M. Janicki, Tudorita Tumbar, Andrew S. Belmont: Visualization of gene activity in living cells . In: Nature Cell Biology . tape 2 , no. December 12 , 2000, ISSN 1465-7392 , pp. 871-878 , doi : 10.1038 / 35046510 ( nature.com [accessed March 24, 2019]).
- Jump up ↑ Salman F. Banani, Allyson M. Rice, William B. Peeples, Yuan Lin, Saumya Jain: Compositional Control of Phase-Separated Cellular Bodies . In: Cell . tape 166 , no. 3 , July 2016, p. 651–663 , doi : 10.1016 / j.cell.2016.06.010 ( elsevier.com [accessed April 5, 2019]).
- ↑ Myriam Scherer, Thomas Stamminger: Emerging Role of PML nuclear bodies in Innate Immune Signaling . In: Journal of Virology . tape 90 , no. 13 , July 1, 2016, ISSN 0022-538X , p. 5850-5854 , doi : 10.1128 / JVI.01979-15 , PMID 27053550 , PMC 4907236 (free full text) - ( asm.org [accessed April 5, 2019]).
- ↑ Inn Chung, Sarah Osterwald, Katharina I. Deeg, Karsten Rippe: PML body meets telomere: The beginning of an ALTernate ending? In: Nucleus . tape 3 , no. 3 , May 2012, ISSN 1949-1034 , p. 263-275 , doi : 10.4161 / nucl.20326 , PMID 22572954 , PMC 3414403 (free full text) - ( tandfonline.com [accessed April 13, 2019]).