Stress Induces Dynamic, Cytotoxicity-Antagonizing TDP-43 Nuclear Bodies via Paraspeckle LncRNA NEAT1-Mediated Liquid-Liquid Phase Separation.

Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.

Gedunin Degrades Aggregates of Mutant Huntingtin Protein and Intranuclear Inclusions via the Proteasomal Pathway in Neurons and Fibroblasts from Patients with Huntington's Disease.

Huntington's disease (HD) is a deadly neurodegenerative disease with abnormal expansion of CAG repeats in the huntingtin gene. Mutant Huntingtin protein (mHTT) forms abnormal aggregates and intranuclear inclusions in specific neurons, resulting in cell death. Here, we tested the ability of a natural heat-shock protein 90 inhibitor, Gedunin, to degrade transfected mHTT in Neuro-2a cells and endogenous mHTT aggregates and intranuclear inclusions in both fibroblasts from HD patients and neurons derived from induced pluripotent stem cells from patients. Our data showed that Gedunin treatment degraded transfected mHTT in Neuro-2a cells, endogenous mHTT aggregates and intranuclear inclusions in fibroblasts from HD patients, and in neurons derived from induced pluripotent stem cells from patients in a dose- and time-dependent manner, and its activity depended on the proteasomal pathway rather than the autophagy route. These findings also showed that although Gedunin degraded abnormal mHTT aggregates and intranuclear inclusions in cells from HD patient, it did not affect normal cells, thus providing a new perspective for using Gedunin to treat HD.

Sigma-1 receptor is involved in degradation of intranuclear inclusions in a cellular model of Huntington's disease.

The sigma-1 receptor (SIGMAR1) is one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins via the ER-related degradation machinery linked to the ubiquitin-proteasome pathway. ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. Recently, we demonstrated that accumulation of SIGMAR1 was common to neuronal nuclear inclusions in polyglutamine diseases including Huntington's disease. Our study also indicated that SIGMAR1 might shuttle between the cytoplasm and the nucleus. In the present study, we investigated the role of SIGMAR1 in nuclear inclusion (NI) formation, using HeLa cells transfected with N-terminal mutant huntingtin. Cell harboring the mutant huntingtin produced SIGMAR1-positive NIs. SIGMAR1 siRNA and a specific inhibitor of the proteasome (epoxomicin) caused significant accumulation of aggregates in the cytoplasm and nucleus. A specific inhibitor of exportin 1 (leptomycin B) also caused NIs. Huntingtin became insolubilized in Western blot analysis after treatments with SIGMAR1 siRNA and epoxomicin. Furthermore, proteasome activity increased chronologically along with the accumulation of mutant huntingtin, but was significantly reduced in cells transfected with SIGMAR1 siRNA. By contrast, overexpression of SIGMAR1 reduced the accumulation of NIs containing mutant huntingtin. Although the LC3-I level was decreased in cells treated with both SIGMAR1 siRNA and control siRNA, the levels of LC3-II and p62 were unchanged. SIGMAR1 agonist and antagonist had no effect on cellular viability and proteasome activity. These findings suggest that the ubiquitin-proteasome pathway is implicated in NI formation, and that SIGMAR1 degrades aberrant proteins in the nucleus via the ER-related degradation machinery. SIGMAR1 might be a promising candidate for therapy of Huntington's disease.

Importance of ERK activation in As2O3-induced differentiation and promyelocytic leukemia nuclear bodies formation in neuroblastoma cells.

Neuroblastoma malignant cell growth is dependent on their undifferentiated status. Arsenic trioxide (As2O3) induces neuroblastoma cell differentiation in vitro, but its mechanisms still remains unknown. We used three human neuroblastoma cell lines (SH-SY5Y, IGR-N-91, LAN-1) that differ from their MYCN and p53 status to explore the intracellular events activated by As2O3 and involved in neurite outgrowth, a morphological marker of differentiation. As2O3 (2µM) induced neurite outgrowth in all cell lines, which was dependent on ERK activation but independent on MYCN status. This process was induced either by a sustained (3 days) or a transient (2h) incubation with As2O3, indicating that very early events trigger the induction of differentiation. In parallel, As2O3 induced a rapid assembly of promyelocytic leukemia nuclear bodies (PML-NB) in an ERK-dependent manner. In conclusion, mechanisms leading to neuroblastoma cell differentiation in response to As2O3 appear to involve the ERK pathway activation and PML-NB formation, which are observed in response to other differentiating molecules such as retinoic acid derivates. This open new perspectives based on the use of treatment combinations to potentiate the differentiating effects of each drug alone and reduce their adverse side effects.

Prolonged treatment with DNMT inhibitors induces distinct effects in promoters and gene-bodies.

Treatment with the demethylating drugs 5-azacytidine (AZA) and decitabine (DAC) is now recognised as an effective therapy for patients with Myelodysplastic Syndromes (MDS), a range of disorders arising in clones of hematopoietic progenitor cells. A variety of cell models have been used to study the effect of these drugs on the methylation of promoter regions of tumour suppressor genes, with recent efforts focusing on the ability of these drugs to inhibit DNA methylation at low doses. However, it is still not clear how nano-molar drug treatment exerts its effects on the methylome. In this study, we have characterised changes in DNA methylation caused by prolonged low-dose treatment in a leukemic cell model (SKM-1), and present a genome-wide analysis of the effects of AZA and DAC. At nano-molar dosages, a one-month continuous treatment halved the total number of hypermethylated probes in leukemic cells and our analysis identified 803 candidate regions with significant demethylation after treatment. Demethylated regions were enriched in promoter sequences whereas gene-body CGIs were more resistant to the demethylation process. CGI methylation in promoters was strongly correlated with gene expression but this correlation was lost after treatment. Our results indicate that CGI demethylation occurs preferentially at promoters, but that it is not generally sufficient to modify expression patterns, and emphasises the roles of other means of maintaining cell state.

Reelin is a target of polyglutamine expanded ataxin-7 in human spinocerebellar ataxia type 7 (SCA7) astrocytes.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative disorder that results from polyglutamine expansion of the ataxin-7 (ATXN7) protein. Remarkably, although mutant ATXN7 is expressed throughout the body, pathology is restricted primarily to the cerebellum and retina. One major goal has been to identify factors that contribute to the tissue specificity of SCA7. Here we describe the development and use of a human astrocyte cell culture model to identify reelin, a factor intimately involved in the development and maintenance of Purkinje cells and the cerebellum as a whole, as an ATXN7 target gene. We found that polyglutamine expansion decreased ATXN7 occupancy, which correlated with increased levels of histone H2B monoubiquitination, at the reelin promoter. Treatment with trichostatin A, but not other histone deacetylase inhibitors, partially restored reelin transcription and promoted the accumulation of mutant ATXN7 into nuclear inclusions. Our findings suggest that reelin could be a previously unknown factor involved in the tissue specificity of SCA7 and that trichostatin A may ameliorate deleterious effects of the mutant ATXN7 protein by promoting its sequestration away from promoters into nuclear inclusions.

Accelerated human mutant tau aggregation by knocking out murine tau in a transgenic mouse model.

Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau(-/-) mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau(-/-) mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies.

In vitro analysis of rod composition and actin dynamics in inherited myopathies.

Rods are the pathological hallmark of nemaline myopathy, but they can also occur as a secondary phenomenon in other disorders, including mitochondrial myopathies such as complex I deficiency. The mechanisms of rod formation are not well understood, particularly when rods occur in diverse disorders with very different structural and metabolic defects. We compared the characteristics of rods associated with abnormalities in structural components of skeletal muscle thin filament (3 mutations in the skeletal actin gene ACTA1) with those of rods induced by the metabolic cell stress of adenosine triphosphate depletion. C2C12 and NIH/3T3 cell culture models and immunocytochemistry were used to study rod composition and conformation. Fluorescent recovery after photobleaching was used to measure actin dynamics inside the rods. We demonstrate that not all rods are the same. Rods formed under different conditions contain a unique fingerprint of actin-binding proteins (cofilin and alpha-actinin) and display differences in actin dynamics that are specific to the mutation, to the cellular location of the rods (intranuclear vs cytoplasmic), and/or to the underlying pathological process (i.e. mutant actin or adenosine triphosphate depletion). Thus, rods likely represent a common morphological end point of a variety of different pathological processes, either structural or metabolic.

B2 attenuates polyglutamine-expanded androgen receptor toxicity in cell and fly models of spinal and bulbar muscular atrophy.

Expanded polyglutamine tracts cause neurodegeneration through a toxic gain-of-function mechanism. Generation of inclusions is a common feature of polyglutamine diseases and other protein misfolding disorders. Inclusion formation is likely to be a defensive response of the cell to the presence of unfolded protein. Recently, the compound B2 has been shown to increase inclusion formation and decrease toxicity of polyglutamine-expanded huntingtin in cultured cells. We explored the effect of B2 on spinal and bulbar muscular atrophy (SBMA). SBMA is caused by expansion of polyglutamine in the androgen receptor (AR) and is characterized by the loss of motor neurons in the brainstem and spinal cord. We found that B2 increases the deposition of mutant AR into nuclear inclusions, without altering the ligand-induced aggregation, expression, or subcellular distribution of the mutant protein. The effect of B2 on inclusions was associated with a decrease in AR transactivation function. We show that B2 reduces mutant AR toxicity in cell and fly models of SBMA, further supporting the idea that accumulation of polyglutamine-expanded protein into inclusions is protective. Our findings suggest B2 as a novel approach to therapy for SBMA.

SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7.

Post-translational modification by SUMO (small ubiquitin-like modifier) was proposed to modulate the pathogenesis of several neurodegenerative diseases. Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder, whose pathology is caused by an expansion of a polyglutamine stretch in the protein ataxin-7 (ATXN7). Here, we identified ATXN7 as new target for SUMOylation in vitro and in vivo. The major SUMO acceptor site was mapped to lysine 257, which is part of an evolutionarily conserved consensus SUMOylation motif. SUMOylation did not influence the subcellular localization of ATXN7 nor its interaction with components of the TFTC/STAGA complex. Expansion of the polyglutamine stretch did not impair the SUMOylation of ATXN7. Furthermore, SUMO1 and SUMO2 colocalized with ATXN7 in a subset of neuronal intranuclear inclusions in the brain of SCA7 patients and SCA7 knock-in mice. In a COS-7 cellular model of SCA7, in addition to diffuse nucleoplasmic staining we identified two populations of nuclear inclusions: homogenous or non-homogenous. Non-homogenous inclusions showed significantly reduced colocalization with SUMO1 and SUMO2, but were highly enriched in Hsp70, 19S proteasome and ubiquitin. Interestingly, they were characterized by increased staining with the apoptotic marker caspase-3 and by disruption of PML nuclear bodies. Importantly, preventing the SUMOylation of expanded ATXN7 by mutating the SUMO site increased both the amount of SDS-insoluble aggregates and of caspase-3 positive non-homogenous inclusions, which act toxic to the cells. Our results demonstrate an influence of SUMOylation on the multistep aggregation process of ATXN7 and implicate a role for ATXN7 SUMOylation in SCA7 pathogenesis.

A novel resveratrol analogue HS-1793 treatment overcomes the resistance conferred by Bcl-2 and is associated with the formation of mature PML nuclear bodies in renal clear cell carcinoma Caki-1 cells.

Bcl-2 protects cancer cells from the apoptotic effects of various chemotherapeutic agents. Inhibition or downregulation of Bcl-2 represents a new therapeutic approach to bypass chemoresistance in cancer cells. Previously we designed and synthesized the resveratrol analogue HS-1793 displaying stronger antitumor efficacy than resveratrol and further demonstrated the HS-1793 resistance conferred by Bcl-2 in human leukemic U937 cells. We undertook this study to determine if HS-1793 treatment can bypass the anti-apoptotic effects of Bcl-2 in human renal cancer cells, with a specific focus on the involvement of promyelocytic leukemia nuclear bodies (PML-NBs). Experiments were conducted with Bcl-2-overexpressing human renal clear cell carcinoma Caki-1 cells. Various apoptosis assessment assays demonstrated that HS-1793 overcomes the resistance conferred by Bcl-2 in Caki-1 cells by inducing apoptosis. We elucidated that HS-1793-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in Caki-1 cells. Our findings show that the resveratrol analogue HS-1793 might provide a novel promising strategy for overcoming the resistance conferred by Bcl-2 via PML protein and the formation of mature PML-NBs.

SAHA treatment overcomes the anti-apoptotic effects of Bcl-2 and is associated with the formation of mature PML nuclear bodies in human leukemic U937 cells.

Bcl-2 protects tumor cells from the apoptotic effects of various antineoplastic agents. Increased expression of Bcl-2 has been associated with poor response to chemotherapy in various malignancies, including leukemia. Therefore, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. We undertook this study to examine whether SAHA (suberoylanilide hydroxamic acid) overcomes the resistance by Bcl-2 in human leukemic cells, with a specific focus on the involvement of PML-NBs. Experiments were conducted with Bcl-2-overexpressing human leukemic U937 cells. Since we previously demonstrated that overexpression of Bcl-2 attenuates resveratrol-induced apoptosis in human leukemic U937 cells, resveratrol-treated U937 cells were used as a negative control. The present study indicates that SAHA at 1-7 microM, the dose range known to induce apoptosis in various cancer cells, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2-overexpressing human leukemic U937 cells. Notably, we observed that SAHA-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in human leukemic U937 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that could help bypass the resistance to apoptosis conferred by Bcl-2. Elucidating exactly how PML regulates Bcl-2 will require further work.

Nanoparticle-induced cell culture models for degenerative protein aggregation diseases.

Protein aggregates and nuclear inclusions containing components of the ubiquitin-proteasome system, expanded polyglutamine (polyQ) proteins, and transcriptional co-activators characterize cellular responses to stress and are hallmarks of neurodegenerative diseases. For instance, in Huntington's disease, an expansion of a polyQ region causes its aggregation into beta-sheet-containing amyloid fibrils. The biological function of polyQ-containing inclusions is unknown. By means of a silica nanoparticle (NP)-based strategy we induced intranuclear protein inclusions that form amyloid-like structures, recapitulating the protein composition and solubility of polyQ-induced nuclear protein aggregates exactly. We showed that global proteasomal proteolysis increases in silica-NP-treated nuclei and, on the local level, a subpopulation of nuclear inclusions overlaps with focal domains of proteasome-dependent protein degradation. The results suggest that inclusions in the nucleus constitute active proteolysis modules that may serve to concentrate and decompose damaged, mal-folded, or misplaced proteins. While nanoparticle-nucleus interactions turn out to be invaluable tools to study the molecular mechanisms of degenerative protein aggregation diseases, one also has to consider the other side of the coin, namely, emerging environmental risks posed by these very interactions.

Puromycin-based vectors promote a ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70 and Proteasomes.

BACKGROUND: Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation. RESULTS: In the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells. CONCLUSION: Taken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design.

Involvement of PML nuclear bodies in CBP degradation through the ubiquitin-proteasome pathway.

Transcriptional coactivator CBP is involved in the regulation of an array of biological processes including cellular differentiation, proliferation and survival. The function of CBP is critical for proper embryonic development and is relevant in cancer biology. Although much is known about the functional roles of CBP in these cellular processes, fewer studies have assessed what in turn regulates CBP activity per se. It has been reported that CBP colocalizes with PML bodies which are nuclear structures disrupted in acute promyelocytic leukemia. However, the biological relevance of CBP localization to PML nuclear bodies is still unclear. In this study, we demonstrate that histone deacetylase inhibitors such as valproic acid, a therapeutically relevant compound used for the treatment of epilepsy, modulates CBP activity. Valproic acid reduces the steady-state level of CBP by inducing CBP degradation through the ubiquitin-proteasome pathway, while increasing the colocalization of CBP with ubiquitin nuclear speckles and with PML nuclear bodies. Our results suggest that PML nuclear bodies are nuclear sites involved in the ubiquitin-dependent degradation of CBP, providing novel insights in the regulation of CBP function and highlighting the relevance of its localization to PML nuclear bodies.

Nuclear organization of PML bodies in leukaemic and multiple myeloma cells.

The nuclear arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) was studied in vitro after the cell treatment by clinically used agents such as all-trans retinoic acid (RA) in human leukaemia and cytostatics or gamma radiation in multiple myeloma cells. In addition, the influence of phorbol ester (PMA) on PML NBs formation was analyzed. A reduced number of PML bodies, which led to relocation of PML NBs closer to the nuclear interior, mostly accompanied RA- and PMA-induced differentiation. Centrally located PML NBs were associated with transcriptional protein RNAP II and SC35 regions, which support importance of PML NBs in RNA processing that mostly proceeds within the nuclear interior. Conversely, the quantity of PML NBs was increased after cytostatic treatment, which caused re-distribution of PML NBs closer to the nuclear envelope. Here we showed correlations between the number of PML NBs and average Centre-to-PML distances. Moreover, a number of cells in S phase, especially during differentiation, influenced number of PML NBs. Studying the proteins involved in PML compartment, such as c-MYC, cell-type specific association of c-MYC and the PML NBs was observed in selected leukaemic cells undergoing differentiation, which was accompanied by c-MYC down-regulation.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Basal, but not overload-induced, myonuclear addition is attenuated by NG-nitro-L-arginine methyl ester (L-NAME) administration.

The purpose of this study was to examine the effect of blocking nitric oxide synthase (NOS) activity via NG-nitro-L-arginine methyl ester (L-NAME) on myonuclear addition in skeletal muscle under basal and overloaded conditions. Female Sprague-Dawley rats (approx. 220 g) were placed into 1 of the following 4 groups (n = 7-9/group): 7-day skeletal muscle overload (O), sham operation (S), skeletal muscle overload with L-NAME treatment (OLN), and sham operation with L-NAME treatment (SLN). Plantaris muscles were overloaded via bilateral surgical ablation of the gastrocnemius muscles and L-NAME (0.75 mg/mL) was administered in the animals' daily drinking water starting 2 days prior to surgery and continued until sacrifice. Myonuclear addition was assessed as subsarcolemmal incorporation of nuclei labeled with 5-bromo-2'-deoxyuridine (approx. 25 mg.(kg body mass)-1.day-1) delivered via osmotic pump during the overload period. As expected, muscle wet mass, total protein content, fiber cross-sectional area, and myonuclear addition were significantly higher (p

Activity-regulated cytoskeleton-associated protein Arc/Arg3.1 binds to spectrin and associates with nuclear promyelocytic leukemia (PML) bodies.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene, whose expression in the central nervous system is induced by specific patterns of synaptic activity. Arc is required for the late-phase of long-term potentiation (LTP) and memory consolidation, and has been implicated in AMPA receptor trafficking. Since Arc's molecular function remains incompletely understood, we have determined its subcellular localization in cultured hippocampal neurons and HEK 293T cells. Fluorescence microscopy experiments revealed that both endogenous and exogenous Arc protein was primarily found in the nucleus, where it concentrated in puncta associated with promyelocytic leukemia (PML) bodies, proposed sites of transcriptional regulation. Arc co-localized and interacted with the betaIV spectrin splice variant betaSpIVSigma5, a nuclear spectrin isoform associated with PML bodies and the nuclear matrix. A small region of Arc containing the coiled-coil domain is also restricted to beta-spectrin-positive puncta, while the isolated spectrin homology domain is diffusely localized. Finally, Arc and betaSpIVSigma5 synergistically increased the number of PML bodies. These results suggest that Arc functions as a spectrin-binding protein, forming a complex that may provide a role at sites of transcriptional regulation within the nucleus.

Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts.

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.