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1.
EMBO Mol Med ; 16(6): 1324-1351, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38730056

RESUMO

Clear-cell renal cell carcinoma (ccRCC), the major subtype of RCC, is frequently diagnosed at late/metastatic stage with 13% 5-year disease-free survival. Functional inactivation of the wild-type p53 protein is implicated in ccRCC therapy resistance, but the detailed mechanisms of p53 malfunction are still poorly characterized. Thus, a better understanding of the mechanisms of disease progression and therapy resistance is required. Here, we report a novel ccRCC dependence on the promyelocytic leukemia (PML) protein. We show that PML is overexpressed in ccRCC and that PML depletion inhibits cell proliferation and relieves pathologic features of anaplastic disease in vivo. Mechanistically, PML loss unleashed p53-dependent cellular senescence thus depicting a novel regulatory axis to limit p53 activity and senescence in ccRCC. Treatment with the FDA-approved PML inhibitor arsenic trioxide induced PML degradation and p53 accumulation and inhibited ccRCC expansion in vitro and in vivo. Therefore, by defining non-oncogene addiction to the PML gene, our work uncovers a novel ccRCC vulnerability and lays the foundation for repurposing an available pharmacological intervention to restore p53 function and chemosensitivity.


Assuntos
Carcinoma de Células Renais , Senescência Celular , Neoplasias Renais , Proteína da Leucemia Promielocítica , Proteína Supressora de Tumor p53 , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/tratamento farmacológico , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Senescência Celular/efeitos dos fármacos , Animais , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Trióxido de Arsênio/farmacologia , Camundongos
2.
Nucleic Acids Res ; 52(11): 6472-6489, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38752489

RESUMO

Orphan nuclear receptors (NRs), such as COUP-TF1, COUP-TF2, EAR2, TR2 and TR4, are implicated in telomerase-negative cancers that maintain their telomeres through the alternative lengthening of telomeres (ALT) mechanism. However, how telomere association of orphan NRs is involved in ALT activation remains unclear. Here, we demonstrate that telomeric tethering of orphan NRs in human fibroblasts initiates formation of ALT-associated PML bodies (APBs) and features of ALT activity, including ALT telomere DNA synthesis, telomere sister chromatid exchange, and telomeric C-circle generation, suggesting de novo ALT induction. Overexpression of orphan NRs exacerbates ALT phenotypes in ALT cells, while their depletion limits ALT. Orphan NRs initiate ALT via the zinc finger protein 827, suggesting the involvement of chromatin structure alterations for ALT activation. Furthermore, we found that orphan NRs and deficiency of the ALT suppressor ATRX-DAXX complex operate in concert to promote ALT activation. Moreover, PML depletion by gene knockout or arsenic trioxide treatment inhibited ALT induction in fibroblasts and ALT cancer cells, suggesting that APB formation underlies the orphan NR-induced ALT activation. Importantly, arsenic trioxide administration abolished APB formation and features of ALT activity in ALT cancer cell line-derived mouse xenografts, suggesting its potential for further therapeutic development to treat ALT cancers.


Assuntos
Fibroblastos , Proteína da Leucemia Promielocítica , Homeostase do Telômero , Humanos , Animais , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/genética , Camundongos , Fibroblastos/metabolismo , Telômero/metabolismo , Telômero/genética , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Troca de Cromátide Irmã , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Trióxido de Arsênio/farmacologia , Chaperonas Moleculares
3.
Commun Biol ; 7(1): 532, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710927

RESUMO

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.


Assuntos
Resposta ao Choque Térmico , Metabolismo dos Lipídeos , Sumoilação , Ubiquitinas , Humanos , Metabolismo dos Lipídeos/genética , Resposta ao Choque Térmico/genética , Regulação da Expressão Gênica , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/genética , Células HeLa , Proteína SUMO-1/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Células HEK293 , Transcrição Gênica , beta Carioferinas/metabolismo , beta Carioferinas/genética
4.
Proc Natl Acad Sci U S A ; 121(18): e2317690121, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38648485

RESUMO

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.


Assuntos
Fator de Transcrição GATA2 , Células-Tronco Hematopoéticas , Proteínas de Fusão Oncogênica , Animais , Humanos , Camundongos , Sítios de Ligação , Autorrenovação Celular , Cromatina/metabolismo , DNA/metabolismo , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA2/genética , Células-Tronco Hematopoéticas/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/genética , Ligação Proteica , Receptor alfa de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico/genética
5.
Cell Death Differ ; 31(6): 768-778, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38627584

RESUMO

The alternative splicing of PML precursor mRNA gives rise to various PML isoforms, yet their expression profile in breast cancer cells remains uncharted. We discovered that PML1 is the most abundant isoform in all breast cancer subtypes, and its expression is associated with unfavorable prognosis in estrogen receptor-positive (ER+) breast cancers. PML depletion reduces cell proliferation, invasion, and stemness, while heterologous PML1 expression augments these processes and fuels tumor growth and resistance to fulvestrant, an FDA-approved drug for ER+ breast cancer, in a mouse model. Moreover, PML1, rather than the well-known tumor suppressor isoform PML4, rescues the proliferation of PML knockdown cells. ChIP-seq analysis reveals significant overlap between PML-, ER-, and Myc-bound promoters, suggesting their coordinated regulation of target gene expression, including genes involved in breast cancer stem cells (BCSCs), such as JAG1, KLF4, YAP1, SNAI1, and MYC. Loss of PML reduces BCSC-related gene expression, and exogenous PML1 expression elevates their expression. Consistently, PML1 restores the association of PML with these promoters in PML-depleted cells. We identified a novel association between PML1 and WDR5, a key component of H3K4 methyltransferase (HMTs) complexes that catalyze H3K4me1 and H3K4me3. ChIP-seq analyses showed that the loss of PML1 reduces H3K4me3 in numerous loci, including BCSC-associated gene promoters. Additionally, PML1, not PML4, re-establishes the H3K4me3 mark on these promoters in PML-depleted cells. Significantly, PML1 is essential for recruiting WDR5, MLL1, and MLL2 to these gene promoters. Inactivating WDR5 by knockdown or inhibitors phenocopies the effects of PML1 loss, reducing BCSC-related gene expression and tumorsphere formation and enhancing fulvestrant's anticancer activity. Our findings challenge the conventional understanding of PML as a tumor suppressor, redefine its role as a promoter of tumor growth in breast cancer, and offer new insights into the unique roles of PML isoforms in breast cancer.


Assuntos
Neoplasias da Mama , Histonas , Fator 4 Semelhante a Kruppel , Células-Tronco Neoplásicas , Proteína da Leucemia Promielocítica , Receptores de Estrogênio , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Feminino , Proteína da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/genética , Animais , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica
6.
Cell Signal ; 119: 111156, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38574938

RESUMO

In the seemingly well-researched field of vascular research, there are still many underestimated factors and molecular mechanisms. In recent years, SUMOylation has become increasingly important. SUMOylation is a post-translational modification in which small ubiquitin-related modifiers (SUMO) are covalently attached to target proteins. Sites where these SUMO modification processes take place in the cell nucleus are PML nuclear bodies (PML-NBs) - multiprotein complexes with their essential main component and organizer, the PML protein. PML and SUMO, either alone or as partners, influence a variety of cellular processes, including regulation of transcription, senescence, DNA damage response and defence against microorganisms, and are involved in innate immunity and inflammatory responses. They also play an important role in maintaining homeostasis in the vascular system and in pathological processes leading to the development and progression of cardiovascular diseases. This review summarizes information about the function of SUMO(ylation) and PML(-NBs) in the human vasculature from angiogenesis to disease and highlights their clinical potential as drug targets.


Assuntos
Proteínas Nucleares , Proteína da Leucemia Promielocítica , Sumoilação , Fatores de Transcrição , Humanos , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Supressoras de Tumor/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia
7.
Nucleus ; 15(1): 2321265, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38411156

RESUMO

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.


Assuntos
Leucemia Promielocítica Aguda , Corpos Nucleares da Leucemia Promielocítica , Humanos , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia
8.
Nucleic Acids Res ; 52(5): 2273-2289, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38118002

RESUMO

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.


Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Antígenos Nucleares , Autoantígenos , Neoplasias Oculares , Histonas , Melanoma , Humanos , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Desmetilação , Metilação de DNA , Histonas/genética , Histonas/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , RNA/metabolismo , Fatores de Transcrição/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Neoplasias Oculares/metabolismo
9.
Viruses ; 15(12)2023 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-38140552

RESUMO

Nuclear bodies (NBs) are dynamic structures present in eukaryotic cell nuclei. They are not bounded by membranes and are often considered biomolecular condensates, defined structurally and functionally by the localisation of core components. Nuclear architecture can be reorganised during normal cellular processes such as the cell cycle as well as in response to cellular stress. Many plant and animal viruses target their proteins to NBs, in some cases triggering their structural disruption and redistribution. Although not all such interactions have been well characterised, subversion of NBs and their functions may form a key part of the life cycle of eukaryotic viruses that require the nucleus for their replication. This review will focus on Cajal bodies (CBs) and the viruses that target them. Since CBs are dynamic structures, other NBs (principally nucleoli and promyelocytic leukaemia, PML and bodies), whose components interact with CBs, will also be considered. As well as providing important insights into key virus-host cell interactions, studies on Cajal and associated NBs may identify novel cellular targets for development of antiviral compounds.


Assuntos
Viroses , Vírus , Animais , Proteínas Nucleares/metabolismo , Corpos Enovelados/metabolismo , Núcleo Celular , Vírus/metabolismo , Viroses/metabolismo , Proteína da Leucemia Promielocítica/metabolismo
10.
Biomolecules ; 13(12)2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38136675

RESUMO

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.


Assuntos
Corpos Nucleares da Leucemia Promielocítica , Proteoma , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/química , Proteína da Leucemia Promielocítica/metabolismo , Proteoma/metabolismo , Prevalência , Separação de Fases , Sumoilação
11.
Virol J ; 20(1): 280, 2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38031162

RESUMO

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.


Assuntos
Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Animais , Suínos , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Fatores de Transcrição/genética , Interferons , Isoformas de Proteínas/genética , Replicação Viral
12.
Nucleic Acids Res ; 51(20): 11024-11039, 2023 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-37823593

RESUMO

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.


Assuntos
Cromatina , Neoplasias de Mama Triplo Negativas , Humanos , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral
13.
Nat Commun ; 14(1): 6111, 2023 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-37777511

RESUMO

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.


Assuntos
Caseína Quinase II , Corpos Nucleares da Leucemia Promielocítica , Proteína da Leucemia Promielocítica , RecQ Helicases , Humanos , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , RecQ Helicases/metabolismo , Ubiquitinação , Sumoilação , Proteína SUMO-1
14.
Structure ; 31(9): 1086-1099.e6, 2023 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-37473756

RESUMO

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.


Assuntos
Proteínas Nucleares , Zinco , Proteínas Nucleares/metabolismo , Zinco/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Sítios de Ligação , Oxirredução
15.
J Virol ; 97(7): e0032823, 2023 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-37338350

RESUMO

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.


Assuntos
Hepatite B , Leucemia , Humanos , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , Antivirais/farmacologia , DNA Viral/genética , DNA Viral/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Replicação Viral/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo
16.
Virol J ; 20(1): 82, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37127643

RESUMO

Promyelocytic leukemia nuclear bodies (PM NBs), often referred to as membraneless organelles, are dynamic macromolecular protein complexes composed of a PML protein core and other transient or permanent components. PML NBs have been shown to play a role in a wide variety of cellular processes. This review describes in detail the diverse and complex interactions between small and medium size DNA viruses and PML NBs that have been described to date. The PML NB components that interact with small and medium size DNA viruses include PML protein isoforms, ATRX/Daxx, Sp100, Sp110, HP1, and p53, among others. Interaction between viruses and components of these NBs can result in different outcomes, such as influencing viral genome expression and/or replication or impacting IFN-mediated or apoptotic cell responses to viral infection. We discuss how PML NB components abrogate the ability of adenoviruses or Hepatitis B virus to transcribe and/or replicate their genomes and how papillomaviruses use PML NBs and their components to promote their propagation. Interactions between polyomaviruses and PML NBs that are poorly understood but nevertheless suggest that the NBs can serve as scaffolds for viral replication or assembly are also presented. Furthermore, complex interactions between the HBx protein of hepadnaviruses and several PML NBs-associated proteins are also described. Finally, current but scarce information regarding the interactions of VP3/apoptin of the avian anellovirus with PML NBs is provided. Despite the considerable number of studies that have investigated the functions of the PML NBs in the context of viral infection, gaps in our understanding of the fine interactions between viruses and the very dynamic PML NBs remain. The complexity of the bodies is undoubtedly a great challenge that needs to be further addressed.


Assuntos
Vírus de DNA , Proteínas Nucleares , Adenoviridae , Proteínas Nucleares/metabolismo , Corpos Nucleares da Leucemia Promielocítica , Proteína da Leucemia Promielocítica/metabolismo , Fatores de Transcrição/metabolismo , Vírus , Vírus de DNA/genética
17.
Viruses ; 15(5)2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37243155

RESUMO

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.


Assuntos
Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Herpesvirus Humano 1/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
18.
Int J Mol Sci ; 24(8)2023 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-37108469

RESUMO

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.


Assuntos
Células Endoteliais , Interleucina-6 , Humanos , Células Endoteliais/metabolismo , Interleucina-6/genética , Interleucina-8 , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Fatores de Transcrição/metabolismo , Lipoproteínas LDL/metabolismo , Proteína Quinase C/metabolismo
19.
Nat Commun ; 14(1): 1840, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-37019904

RESUMO

Cellular senescence contributes to tissue homeostasis and age-related pathologies. However, how senescence is initiated in stressed cells remains vague. Here, we discover that exposure to irradiation, oxidative or inflammatory stressors induces transient biogenesis of primary cilia, which are then used by stressed cells to communicate with the promyelocytic leukemia nuclear bodies (PML-NBs) to initiate senescence responses in human cells. Mechanistically, a ciliary ARL13B-ARL3 GTPase cascade negatively regulates the association of transition fiber protein FBF1 and SUMO-conjugating enzyme UBC9. Irreparable stresses downregulate the ciliary ARLs and release UBC9 to SUMOylate FBF1 at the ciliary base. SUMOylated FBF1 then translocates to PML-NBs to promote PML-NB biogenesis and PML-NB-dependent senescence initiation. Remarkably, Fbf1 ablation effectively subdues global senescence burden and prevents associated health decline in irradiation-treated mice. Collectively, our findings assign the primary cilium a key role in senescence induction in mammalian cells and, also, a promising target in future senotherapy strategies.


Assuntos
Cílios , Proteínas Nucleares , Humanos , Animais , Camundongos , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Nucleares/metabolismo , Cílios/metabolismo , Corpos Nucleares da Leucemia Promielocítica , Sumoilação , Núcleo Celular/metabolismo , Mamíferos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
20.
Nucleic Acids Res ; 51(7): 3185-3204, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-36912092

RESUMO

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.


Assuntos
Gnathostoma , Retroelementos , Animais , Humanos , Mamíferos/genética , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Isoformas de Proteínas/genética , Retroelementos/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Gnathostoma/enzimologia , Gnathostoma/genética , Gnathostoma/metabolismo
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