SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation

1999 ◽  
Vol 112 (3) ◽  
pp. 381-393 ◽  
Author(s):  
E. Duprez ◽  
A.J. Saurin ◽  
J.M. Desterro ◽  
V. Lallemand-Breitenbach ◽  
K. Howe ◽  
...  
Keyword(s):  
Acute Promyelocytic Leukaemia ◽  
Consensus Sequence ◽  
Nuclear Bodies ◽  
Promyelocytic Leukaemia ◽  
Fusion Product ◽  
Nuclear Localisation ◽  
Target Proteins ◽  
Pml Nuclear Bodies

PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), which are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, we have identified the primary PML-SUMO-1 modification site as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation as only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1. We show that SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. Our results provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APL disease state.

scholarly journals Demonstration of a RNA-dependent nuclear interaction between the promyelocytic leukaemia protein and glyceraldehyde-3-phosphate dehydrogenase

1998 ◽  
Vol 335 (3) ◽  
pp. 691-696 ◽  
Author(s):  
Graeme W. CARLILE ◽  
William G. TATTON ◽  
Katherine L. B. BORDEN
Keyword(s):  
Acute Promyelocytic Leukaemia ◽  
Neuronal Death ◽  
Nuclear Interaction ◽  
Nuclear Bodies ◽  
Promyelocytic Leukaemia ◽  
Pml Nuclear Bodies ◽  
Pml Bodies ◽  
Rnase Treatment ◽  
Rna Disruption ◽  
Apoptotic Neuronal Death

The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes with PML in nuclear bodies. RNase treatment disrupts the ability of PML and GAPDH to both co-localize and co-immunoprecipitate, indicating that the association between PML and GAPDH depends on the presence of RNA. Disruption of PML bodies contributes towards reduced apoptosis in acute promyelocytic leukaemia and GAPDH induces apoptotic neuronal death. The GAPDH–PML interaction may be involved in the regulation of apoptosis.

Trivalent Antimonials Induce Degradation of the PML-RAR Oncoprotein and Reorganization of the Promyelocytic Leukemia Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4308-4316 ◽  
Author(s):  
Stefan Müller ◽  
Wilson H. Miller ◽  
Anne Dejean
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Antimony Trioxide ◽  
Nb4 Cells ◽  
Pml Nuclear Bodies

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor  (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.

scholarly journals Synergy against PML-RARa: targeting transcription, proteolysis, differentiation, and self-renewal in acute promyelocytic leukemia

2013 ◽  
Vol 210 (13) ◽  
pp. 2793-2802 ◽  
Author(s):  
Guilherme Augusto dos Santos ◽  
Lev Kats ◽  
Pier Paolo Pandolfi
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Target Genes ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Pml Nuclear Bodies ◽  
Molecular Events

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by a chimeric oncoprotein containing the C terminus of the retinoic acid receptor-a (RARa) fused to an N-terminal partner, most commonly promyelocytic leukemia protein (PML). Mechanistically, PML-RARa acts as a transcriptional repressor of RARa and non-RARa target genes and antagonizes the formation and function of PML nuclear bodies that regulate numerous signaling pathways. The empirical discoveries that PML-RARa–associated APL is sensitive to both all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO), and the subsequent understanding of the mechanisms of action of these drugs, have led to efforts to understand the contribution of molecular events to APL cell differentiation, leukemia-initiating cell (LIC) clearance, and disease eradication in vitro and in vivo. Critically, the mechanistic insights gleaned from these studies have resulted not only in a better understanding of APL itself, but also carry valuable lessons for other malignancies.

scholarly journals Interaction of the Adenovirus Type 5 E4 Orf3 Protein with Promyelocytic Leukemia Protein Isoform II Is Required for ND10 Disruption

2006 ◽  
Vol 80 (6) ◽  
pp. 3042-3049 ◽  
Author(s):  
Anne Hoppe ◽  
Stephanie J. Beech ◽  
John Dimmock ◽  
Keith N. Leppard
Keyword(s):  
Adenovirus Type ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Promyelocytic Leukemia Protein ◽  
Orf3 Protein ◽  
Pml Nuclear Bodies ◽  
Nuclear Tracks ◽  
Nuclear Structures

ABSTRACT Nuclear domain 10 (ND10s), or promyelocytic leukemia protein (PML) nuclear bodies, are spherical nuclear structures that require PML proteins for their formation. Many viruses target these structures during infection. The E4 Orf3 protein of adenovirus 5 (Ad5) rearranges ND10s, causing PML to colocalize with Orf3 in nuclear tracks or fibers. There are six different PML isoforms (I to VI) present at ND10s, all sharing a common N terminus but with structural differences at their C termini. In this study, PML II was the only one of these six isoforms that was found to interact directly and specifically with Ad5 E4 Orf3 in vitro and in vivo; these results define a new Orf3 activity. Three of a series of 18 mutant Orf3 proteins were unable to interact with PML II; these were also unable to cause ND10 rearrangement. Moreover, in PML-null cells that contained neoformed ND10s comprising a single PML isoform, only ND10s formed of PML II were rearranged by Orf3. These data show that the interaction between Orf3 and PML II is necessary for ND10 rearrangement to occur. Finally, Orf3 was shown to self-associate in vitro. This activity was absent in mutant Orf3 proteins that were unable to form tracks and to bind PML II. Thus, Orf3 oligomerization may mediate the formation of nuclear tracks in vivo and may also be important for PML II binding.

Trivalent Antimonials Induce Degradation of the PML-RAR Oncoprotein and Reorganization of the Promyelocytic Leukemia Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4308-4316 ◽  
Author(s):  
Stefan Müller ◽  
Wilson H. Miller ◽  
Anne Dejean
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Antimony Trioxide ◽  
Nb4 Cells ◽  
Pml Nuclear Bodies

Abstract Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor  (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

scholarly journals Solvent Exposure and Ionic Condensation Drive Fuzzy Dimerization of Disordered Heterochromatin Protein Sequence

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 915
Author(s):  
Jazelli Mueterthies ◽  
Davit A. Potoyan
Keyword(s):  
Phase Separation ◽  
Low Complexity ◽  
Nuclear Bodies ◽  
Disordered Proteins ◽  
Solvent Exposure ◽  
Ion Release ◽  
Dimer Formation ◽  
Eukaryotic Organisms

Proteins with low complexity, disordered sequences are receiving increasing attention due to their central roles in the biogenesis and regulation of membraneless organelles. In eukaryotic organisms, a substantial fraction of disordered proteins reside in the nucleus, thereby facilitating the formation of nuclear bodies, nucleolus, and chromatin compartmentalization. The heterochromatin family of proteins (HP1) is an important player in driving the formation of gene silenced mesoscopic heterochromatin B compartments and pericentric regions. Recent experiments have shown that the HP1a sequence of Drosophila melanogaster can undergo liquid-liquid phase separation under both in vitro and in vivo conditions, induced by changes of the monovalent salt concentration. While the phase separation of HP1a is thought to be the mechanism underlying chromatin compartmentalization, the molecular level mechanistic picture of salt-driven phase separation of HP1a has remained poorly understood. The disordered hinge region of HP1a is seen as the driver of salt-induced condensation because of its charge enriched sequence and post-translational modifications. Here, we set out to decipher the mechanisms of salt-induced condensation of HP1a through a systematic study of salt-dependent conformations of single chains and fuzzy dimers of disordered HP1a hinge sequences. Using multiple independent all-atom simulations with and without enhanced sampling, we carry out detailed characterization of conformational ensembles of disordered HP1a chains under different ionic conditions using various polymeric and structural measures. We show that the mobile ion release, enhancement of local transient secondary structural elements, and side-chain exposure to solvent are robust trends that accompany fuzzy dimer formation. Furthermore, we find that salt-induced changes in the ensemble of conformations of HP1a disordered hinge sequence fine-tune the inter-chain vs. self-chain interactions in ways that favor fuzzy dimer formation under low salt conditions in the agreement with condensation trends seen in experiments.

scholarly journals Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence

Neoplasia ◽  
2001 ◽  
Vol 3 (3) ◽  
pp. 179-188 ◽  
Author(s):  
Cynthia M. Simbulan-Rosenthal ◽  
Dean S. Rosenthal ◽  
RuiBai Luo ◽  
Raed Samara ◽  
Mira Jung ◽  
...  
Keyword(s):  
Consensus Sequence

scholarly journals Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

1989 ◽  
Vol 9 (11) ◽  
pp. 5073-5080 ◽  
Author(s):  
M Kozak
Keyword(s):  
Context Effects ◽  
Consensus Sequence ◽  
Negative Regulator ◽  
Late Event ◽  
Free Translation ◽  
Initiator Codon ◽  
Translation Systems ◽  
Short Versions

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters

1993 ◽  
Vol 13 (9) ◽  
pp. 5710-5724
Author(s):  
E DesJardins ◽  
N Hay
Keyword(s):  
Zinc Finger ◽  
Tandem Repeats ◽  
Zinc Finger Protein ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Maximal Activity ◽  
Single Copy ◽  
Promoter Usage

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.

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