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1.
Commun Biol ; 7(1): 532, 2024 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-38710927

RESUMEN

Golgin tethers are known to mediate vesicular transport in the secretory pathway, whereas it is relatively unknown whether they may mediate cellular stress response within the cell. Here, we describe a cellular stress response during heat shock stress via SUMOylation of a Golgin tether, Golgin45. We found that Golgin45 is a SUMOylated Golgin via SUMO1 under steady state condition. Upon heat shock stress, the Golgin enters the nucleus by interacting with Importin-ß2 and gets further modified by SUMO3. Importantly, SUMOylated Golgin45 appears to interact with PML and SUMO-deficient Golgin45 mutant functions as a dominant negative for PML-NB formation during heat shock stress, suppressing transcription of lipid metabolism genes. These results indicate that Golgin45 may play a role in heat stress response by transcriptional regulation of lipid metabolism genes in SUMOylation-dependent fashion.


Asunto(s)
Respuesta al Choque Térmico , Metabolismo de los Lípidos , Sumoilación , Ubiquitinas , Humanos , Metabolismo de los Lípidos/genética , Respuesta al Choque Térmico/genética , Regulación de la Expresión Génica , Proteína de la Leucemia Promielocítica/metabolismo , Proteína de la Leucemia Promielocítica/genética , Células HeLa , Proteína SUMO-1/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Células HEK293 , Transcripción Genética , beta Carioferinas/metabolismo , beta Carioferinas/genética
2.
Proc Natl Acad Sci U S A ; 121(18): e2317690121, 2024 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-38648485

RESUMEN

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.


Asunto(s)
Factor de Transcripción GATA2 , Células Madre Hematopoyéticas , Proteínas de Fusión Oncogénica , Animales , Humanos , Ratones , Sitios de Unión , Autorrenovación de las Células , Cromatina/metabolismo , ADN/metabolismo , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA2/genética , Células Madre Hematopoyéticas/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Proteína de la Leucemia Promielocítica/genética , Unión Proteica , Receptor alfa de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico/genética
3.
Nucleus ; 15(1): 2321265, 2024 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38411156

RESUMEN

Promyelocytic leukemia (PML) nuclear bodies, membrane-less organelles in the nucleus, play a crucial role in cellular homeostasis. These dynamic structures result from the assembly of scaffolding PML proteins and various partners. Recent crystal structure analyses revealed essential self-interacting domains, while liquid-liquid phase separation contributes to their formation. PML bodies orchestrate post-translational modifications, particularly stress-induced SUMOylation, impacting target protein functions. Serving as hubs in multiple signaling pathways, they influence cellular processes like senescence. Dysregulation of PML expression contributes to diseases, including cancer, highlighting their significance. Therapeutically, PML bodies are promising targets, exemplified by successful acute promyelocytic leukemia treatment with arsenic trioxide and retinoic acid restoring PML bodies. Understanding their functions illuminates both normal and pathological cellular physiology, guiding potential therapies. This review explores recent advancements in PML body biogenesis, biochemical activity, and their evolving biological roles.


Asunto(s)
Leucemia Promielocítica Aguda , Cuerpos Nucleares de la Leucemia Promielocítica , Humanos , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología
4.
Nucleic Acids Res ; 52(5): 2273-2289, 2024 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-38118002

RESUMEN

Albeit N1-Methyladenosine (m1A) RNA modification represents an important regulator of RNA metabolism, the role of m1A modification in carcinogenesis remains enigmatic. Herein, we found that histone lactylation enhances ALKBH3 expression and simultaneously attenuates the formation of tumor-suppressive promyelocytic leukemia protein (PML) condensates by removing the m1A methylation of SP100A, promoting the malignant transformation of cancers. First, ALKBH3 is specifically upregulated in high-risk ocular melanoma due to excessive histone lactylation levels, referring to m1A hypomethylation status. Moreover, the multiomics analysis subsequently identified that SP100A, a core component for PML bodies, serves as a downstream candidate target for ALKBH3. Therapeutically, the silencing of ALKBH3 exhibits efficient therapeutic efficacy in melanoma both in vitro and in vivo, which could be reversed by the depletion of SP100A. Mechanistically, we found that YTHDF1 is responsible for recognition of the m1A methylated SP100A transcript, which increases its RNA stability and translational efficacy. Conclusively, we initially demonstrated that m1A modification is necessary for tumor suppressor gene expression, expanding the current understandings of dynamic m1A function during tumor progression. In addition, our results indicate that lactylation-driven ALKBH3 is essential for the formation of PML nuclear condensates, which bridges our knowledge of m1A modification, metabolic reprogramming, and phase-separation events.


Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Antígenos Nucleares , Autoantígenos , Neoplasias del Ojo , Histonas , Melanoma , Humanos , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Desmetilación , Metilación de ADN , Histonas/genética , Histonas/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , ARN/metabolismo , Factores de Transcripción/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Neoplasias del Ojo/metabolismo
5.
Int J Mol Sci ; 24(23)2023 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-38069029

RESUMEN

PML bodies are subnuclear protein complexes that play a crucial role in various physiological and pathological cellular processes. One of the general structural proteins of PML bodies is a member of the tripartite motif (TRIM) family-promyelocytic leukemia protein (PML). It is known that PML interacts with over a hundred partners, and the protein itself is represented by several major isoforms, differing in their variable and disordered C-terminal end due to alternative splicing. Despite nearly 30 years of research, the mechanisms underlying PML body formation and the role of PML proteins in this process remain largely unclear. In this review, we examine the literature and highlight recent progress in this field, with a particular focus on understanding the role of individual domains of the PML protein, its post-translational modifications, and polyvalent nonspecific interactions in the formation of PML bodies. Additionally, based on the available literature, we propose a new hypothetical model of PML body formation.


Asunto(s)
Proteínas Nucleares , Cuerpos Nucleares de la Leucemia Promielocítica , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/química , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos
6.
Biomolecules ; 13(12)2023 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-38136675

RESUMEN

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.


Asunto(s)
Cuerpos Nucleares de la Leucemia Promielocítica , Proteoma , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/química , Proteína de la Leucemia Promielocítica/metabolismo , Proteoma/metabolismo , Prevalencia , Separación de Fases , Sumoilación
7.
Virol J ; 20(1): 280, 2023 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-38031162

RESUMEN

BACKGROUND: Promyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection. METHODS: PML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-ß signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays. RESULTS: Our results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-ß signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection. CONCLUSIONS: These findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-ß and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.


Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Animales , Porcinos , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Factores de Transcripción/genética , Interferones , Isoformas de Proteínas/genética , Replicación Viral
8.
Nucleic Acids Res ; 51(20): 11024-11039, 2023 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-37823593

RESUMEN

The promyelocytic leukemia (PML) protein organizes nuclear aggregates known as PML nuclear bodies (PML-NBs), where many transcription factors localize to be regulated. In addition, associations of PML and PML-NBs with chromatin are described in various cell types, further implicating PML in transcriptional regulation. However, a complete understanding of the functional consequences of PML association to DNA in cellular contexts where it promotes relevant phenotypes is still lacking. We examined PML chromatin association in triple-negative breast cancer (TNBC) cell lines, where it exerts important oncogenic functions. We find that PML associates discontinuously with large heterochromatic PML-associated domains (PADs) that contain discrete gene-rich euchromatic sub-domains locally depleted of PML. PML promotes heterochromatic organization in PADs and expression of pro-metastatic genes embedded in these sub-domains. Importantly, this occurs outside PML-NBs, suggesting that nucleoplasmic PML exerts a relevant gene regulatory function. We also find that PML plays indirect regulatory roles in TNBC cells by promoting the expression of pro-metastatic genes outside PADs. Our findings suggest that PML is an important transcriptional regulator of pro-oncogenic metagenes in TNBC cells, via transcriptional regulation and epigenetic organization of heterochromatin domains that embed regions of local transcriptional activity.


Asunto(s)
Cromatina , Neoplasias de la Mama Triple Negativas , Humanos , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Epigénesis Genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral
9.
Nat Commun ; 14(1): 6111, 2023 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-37777511

RESUMEN

The Boom syndrome helicase (BLM) unwinds a variety of DNA structures such as Guanine (G)-quadruplex. Here we reveal a role of RNF111/Arkadia and its paralog ARKL1, as well as Promyelocytic Leukemia Nuclear Bodies (PML NBs), in the regulation of ubiquitination and control of BLM protein levels. RNF111 exhibits a non-canonical SUMO targeted E3 ligase (STUBL) activity targeting BLM ubiquitination in PML NBs. ARKL1 promotes RNF111 localization to PML NBs through SUMO-interacting motif (SIM) interaction with SUMOylated RNF111, which is regulated by casein kinase 2 (CK2) phosphorylation of ARKL1 at a serine residue near the ARKL1 SIM domain. Upregulated BLM in ARKL1 or RNF111-deficient cells leads to a decrease of G-quadruplex levels in the nucleus. These results demonstrate that a CK2- and RNF111-ARKL1-dependent regulation of BLM in PML NBs plays a critical role in controlling BLM protein levels for the regulation of G-quadruplex.


Asunto(s)
Quinasa de la Caseína II , Cuerpos Nucleares de la Leucemia Promielocítica , Proteína de la Leucemia Promielocítica , RecQ Helicasas , Humanos , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , RecQ Helicasas/metabolismo , Ubiquitinación , Sumoilación , Proteína SUMO-1
10.
Mol Oncol ; 17(10): 2090-2108, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37518985

RESUMEN

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with the suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed the induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth, and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.


Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Proteína de la Leucemia Promielocítica/genética , Factor A de Crecimiento Endotelial Vascular , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Factores de Transcripción/genética , Movimiento Celular
11.
Structure ; 31(9): 1086-1099.e6, 2023 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-37473756

RESUMEN

The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using in vitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the in vitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.


Asunto(s)
Proteínas Nucleares , Zinc , Proteínas Nucleares/metabolismo , Zinc/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Sitios de Unión , Oxidación-Reducción
12.
J Virol ; 97(7): e0032823, 2023 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-37338350

RESUMEN

Hepatitis B virus (HBV) chronically infects approximately 300 million people worldwide, and permanently repressing transcription of covalently closed circular DNA (cccDNA), the episomal viral DNA reservoir, is an attractive approach toward curing HBV. However, the mechanism underlying cccDNA transcription is only partially understood. In this study, by illuminating cccDNA of wild-type HBV (HBV-WT) and transcriptionally inactive HBV that bears a deficient HBV X gene (HBV-ΔX), we found that the HBV-ΔX cccDNA more frequently colocalizes with promyelocytic leukemia (PML) bodies than that of HBV-WT cccDNA. A small interfering RNA (siRNA) screen targeting 91 PML body-related proteins identified SMC5-SMC6 localization factor 2 (SLF2) as a host restriction factor of cccDNA transcription, and subsequent studies showed that SLF2 mediates HBV cccDNA entrapment in PML bodies by interacting with the SMC5/6 complex. We further showed that the region of SLF2 comprising residues 590 to 710 interacts with and recruits the SMC5/6 complex to PML bodies, and the C-terminal domain of SLF2 containing this region is necessary for repression of cccDNA transcription. Our findings shed new light on cellular mechanisms that inhibit HBV infection and lend further support for targeting the HBx pathway to repress HBV activity. IMPORTANCE Chronic HBV infection remains a major public health problem worldwide. Current antiviral treatments rarely cure the infection, as they cannot clear the viral reservoir, cccDNA, in the nucleus. Therefore, permanently silencing HBV cccDNA transcription represents a promising approach for a cure of HBV infection. Our study provides new insights into the cellular mechanisms that restrict HBV infection, revealing the role of SLF2 in directing HBV cccDNA to PML bodies for transcriptional repression. These findings have important implications for the development of antiviral therapies against HBV.


Asunto(s)
Hepatitis B , Leucemia , Humanos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , ADN Circular/genética , ADN Circular/metabolismo , Antivirales/farmacología , ADN Viral/genética , ADN Viral/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Replicación Viral/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo
13.
Viruses ; 15(5)2023 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-37243155

RESUMEN

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 (SIM362-364) 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.


Asunto(s)
Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Herpesvirus Humano 1/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
14.
Int J Mol Sci ; 24(8)2023 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-37108469

RESUMEN

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.


Asunto(s)
Células Endoteliales , Interleucina-6 , Humanos , Células Endoteliales/metabolismo , Interleucina-6/genética , Interleucina-8 , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Factores de Transcripción/metabolismo , Lipoproteínas LDL/metabolismo , Proteína Quinasa C/metabolismo
15.
Nucleic Acids Res ; 51(7): 3185-3204, 2023 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-36912092

RESUMEN

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.


Asunto(s)
Gnathostoma , Retroelementos , Animales , Humanos , Mamíferos/genética , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Isoformas de Proteínas/genética , Retroelementos/genética , Factores de Transcripción/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Gnathostoma/enzimología , Gnathostoma/genética , Gnathostoma/metabolismo
16.
EMBO J ; 42(3): e112058, 2023 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-36524443

RESUMEN

TRIM33 is a chromatin reader required for mammalian mesendoderm differentiation after activation of Nodal signaling, while its role in mESCs is still elusive. Here, we report that TRIM33 co-localizes with promyelocytic leukemia nuclear bodies (PML-NBs) specifically in mESCs, to mediate Nodal signaling-directed transcription of Lefty1/2. We show that TRIM33 puncta formation in mESCs depends on PML and on specific assembly of PML-NBs. Moreover, TRIM33 and PML co-regulate Lefty1/2 expression in mESCs, with both PML protein and formation of mESCs-specific PML-NBs being required for TRIM33 recruitment to these loci, and PML-NBs directly associating with the Lefty1/2 loci. Finally, a TurboID proximity-labeling experiment confirmed that TRIM33 is highly enriched only in mESCs-specific PML-NBs. Thus, our study supports a model in which TRIM33 condensates regulate Nodal signaling-directed transcription in mESCs and shows that PML-NBs can recruit distinct sets of client proteins in a cell-context-dependent manner.


Asunto(s)
Células Madre Embrionarias de Ratones , Cuerpos Nucleares de la Leucemia Promielocítica , Animales , Humanos , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal , Núcleo Celular/metabolismo , Mamíferos , Factores de Transcripción/genética
17.
Acta Haematol ; 146(1): 58-64, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36198282

RESUMEN

More than 95% of patients with acute promyelocytic leukemia (APL) are characterized by the reciprocal translocation t(15;17)(q24;21), which involves the promyelocytic leukemia protein (PML) gene on chromosome 15 and the retinoic acid receptor-α (RARA) gene on chromosome 17, leading to the production of the PML::RARA chimeric gene. Additional chromosomal abnormalities are described in all acute myeloid leukemias and occur in approximately one-third of patients with newly diagnosed APL. Here, we report the case of de novo APL showing the classical t(15;17)(q24;q21), a deletion of the short arm of chromosome 6 (6p), and a noncanonical molecular variant of the PML::RARA transcript. Nevertheless, the patient achieved complete remission after treatment with conventional therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Notwithstanding that the molecular pathogenesis of this type of atypical variant still remains unknown, we conclude that this atypical PML::RARA bcr2 fusion gene associated with del(6p) does not seem to alter the effectiveness of combined treatment with ATRA and ATO.


Asunto(s)
Leucemia Promielocítica Aguda , Humanos , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Tretinoina/uso terapéutico , Proteína de la Leucemia Promielocítica/genética , Proteínas de Fusión Oncogénica/genética
18.
Cell Death Dis ; 13(12): 1042, 2022 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-36522328

RESUMEN

Nuclear actin participates in a continuously expanding list of core processes within eukaryotic nuclei, including the maintenance of genomic integrity. In response to DNA damage, nuclear actin polymerises into filaments that are involved in the repair of damaged DNA through incompletely defined mechanisms. We present data to show that the formation of nuclear F-actin in response to genotoxic stress acts as a scaffold for PML NBs and that these filamentous networks are essential for PML NB fission and recruitment of microbodies to DNA lesions. Further to this, we demonstrate that the accumulation of the toxic lamin A precursor prelamin A induces mislocalisation of nuclear actin to the nuclear envelope and prevents the establishment of nucleoplasmic F-actin networks in response to stress. Consequently, PML NB dynamics and recruitment to DNA lesions is ablated, resulting in impaired DNA damage repair. Inhibition of nuclear export of formin mDia2 restores nuclear F-actin formation by augmenting polymerisation of nuclear actin in response to stress and rescues PML NB localisation to sites of DNA repair, leading to reduced levels of DNA damage.


Asunto(s)
Actinas , Proteínas Nucleares , Actinas/genética , Proteínas Nucleares/genética , Cuerpos Nucleares de la Leucemia Promielocítica , Núcleo Celular , Daño del ADN , ADN , Proteína de la Leucemia Promielocítica/genética
19.
Int J Biol Macromol ; 223(Pt A): 468-478, 2022 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-36356867

RESUMEN

RING-B box-coiled coil (RBCC) domain of promyelocytic leukemia (PML) comprises a zinc finger motif that is targeted by arsenic trioxide (ATO) to treat acute promyelocytic leukemia (APL) pathogenesis. Preliminary evidence suggests that the PML-RBCC has different functional characteristics, but no structural details have been reported despite its importance in differential expression and cell-cycle regulation. Therefore, the recombinant h-PML-RBCC protein was purified to its homogeneity, and characterized for oligomeric behaviour which indicated that RBCC domain exists as a tetramer in solution. Furthermore, nano-DSF and circular-dichroism demonstrated that the tetrameric form preserves its native conformation along with thermal stability (Tm = 83.2 °C). In-silico-based PML-RBCC structure was used to perform the molecular dynamics simulation for 300 ns in the presence of zinc atoms, which demonstrated the differential dynamic of PML-RBCC tetrameric chains. MMPBSA analysis also indicated the role of hydrophobic interactions that favours stable tetrameric structure of PML-RBCC. ATO-induced secondary and tertiary structure changes were observed in PML-RBCC using circular dichroism and fluorescence spectroscopy. Dynamic light scattering and transmission electron microscopy revealed ATO-induced higher-order oligomerization and aggregation of PML-RBCC. The unique oligomeric nature of the h-PML-RBCC protein and its interactions with ATO will help to understand the mechanism of APL pathogenesis and drug resistance.


Asunto(s)
Leucemia Promielocítica Aguda , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Trióxido de Arsénico/uso terapéutico , Proteína de la Leucemia Promielocítica/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
20.
Int J Mol Sci ; 23(19)2022 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-36233232

RESUMEN

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.


Asunto(s)
Infecciones por Citomegalovirus , Infecciones por Herpesviridae , Proteína de la Leucemia Promielocítica , Antivirales/metabolismo , Citomegalovirus , Células Endoteliales/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Replicación Viral
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