Eukaryotic nuclei are subdivided into subnuclear structures. Among the most prominent of these structures are the nucleolus and the PML nuclear bodies (PML‐NBs). PML‐NBs are spherical multiprotein ...aggregates of varying size localized in the interchromosomal area. PML‐NB formation is dependent on the presence of the promyelocytic leukemia protein (PML) as well as on post‐translational modification of core components by covalent attachment of the small ubiquitin‐like modifier (SUMO). So far, PML‐NBs as well as PML have been described in mammalian cells only, whereas no orthologs are known in the plant kingdom. In order to investigate conserved mechanisms in PML targeting, we expressed human PML (hPML) fused to the GFP in Nicotiana benthamiana. Using confocal laser scanning microscopy and coimmunoprecipitation followed by mass spectrometric analysis, we found the fusion protein in association with nucleolar constituents. Importantly, mutants of hPML, which are no longer SUMOylated, showed altered localizations, implying SUMO‐dependent targeting of hPML in plants as has previously been shown for mammalian cells. Interestingly, in the presence of proteasome inhibitors, hPML could also be found in the nucleolus of mammalian cells suggesting conserved targeting mechanisms of PML across kingdoms. Finally, Solanum tuberosum COP1, a proposed PML‐like protein from plants, was fused to the red fluorescent protein (RFP) and coexpressed with hPML::eGFP. Microscopic analysis confirmed the localization of COP1::RFP in nuclear speckles. However, hPML::eGFP did not colocalize with COP1::RFP. Hence, we conclude that plants do not possess specialized PML‐NBs, but that their functions may be covered by other subnuclear structures like the nucleolus.
Database Proteomics data have been deposited to the ProteomeXchange Consortium with the identifier PXD004254.
The signature protein of PML nuclear bodies, human promyelocytic leukemia protein PML, was expressed in Nicotiana benthamiana to study whether these structures are also present in plant cells. Diverging subnuclear localization but similar response to altered SUMOylation suggests that PML nuclear body functions may be covered by other subnuclear structures in plants.
PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family ...of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to mutational escape.
Protection against viruses is provided by the innate, adaptive, and cellular intrinsic immunity. Recently, a subnuclear structure known as nuclear domain 10 (ND10), which represents accumulations of ...the cellular proteins PML, hDaxx, and Sp100, was shown to mediate intrinsic resistance against infections with human cytomegalovirus (HCMV). The immediate-early protein IE1 of HCMV efficiently antagonizes this antiviral defense mechanism by inducing disruption of the ND10 structures. It has been proposed that this event involves de SUMOylation of the PML protein by IE1, since SUMO-modified forms of PML are essential for ND10 formation. The exact mechanism, however, remains unclear.In order to address the question whether IE1 affects additional proteins besides PML to disrupt ND10 integrity, primary human fibroblasts stably expressing FLAG-tagged versions of SUMO-1 or SUMO-3 were generated. Expression of IE1 in these cells, followed by Western blot analyses, revealed that not only PML but also Sp100 is a specific target of IE1-mediated de SUMOylation. Furthermore, it was observed that overexpression of FLAG-SUMO-1 and FLAG-SUMO-3 promotes HCMV replication. These findings indicate that, besides de-SUMOylation of cellular proteins, HCMV has evolved additional mechanisms to manipulate the SUMO conjugation pathway for its own benefit.In order to gain insight into the structural basis of ND10 disruption, determination of the three-dimensional structure of IE1 was performed. The crystal structure of a central, 45 kDa subdomain of the rhesus macaque cytomegalovirus (RhCMV) IE1 protein revealed an unusual all alpha-helical fold that displays no homology to known protein structures. In vitro and in vivo approaches demonstrated that function and structure are conserved between the IE1 proteins of HCMV and RhCMV. Infection experiments with a recombinant HCMV, expressing the truncated IE1 variant, revealed that the IE1 subdomain is sufficient to induce de-SUMOylation of PML. Intruigingly, this resulted in the dispersal of ND10-associated proteins but not of PML itself, suggesting that ND10 disruption by IE1 is an at least two-step process mediated by distinct domains of the IE1 protein. Finally, the crystal structure of IE1 allowed for the generation and initial characterization of mutant IE1 proteins and, thus, represents a valuable tool to further elucidate the mechanism by which IE1 counteracts ND10-based intrinsic immunity.
To determine the frequency of use of biologic DMARDs (bDMARDs) in monotherapy, to describe the baseline characteristics of patients treated with bDMARDs in monotherapy and to compare the ...effectiveness of bDMARDs in monotherapy with that of bDMARDs in combination with synthetic DMARDs (sDMARDs).
Using data from the Swiss RA (SCQM-RA) registry, bDMARD treatment courses (TCs) were classified either as monotherapy or as combination therapy, depending on the presence of concomitant sDMARDs. Prescription of bDMARD monotherapy was analysed using logistic regression. bDMARD retention was analysed using Kaplan-Meier and Cox models with the addition of time-varying covariate effects. Evolution of the DAS28 over time was analysed with mixed-effects models for longitudinal data.
A total of 4218 TCs on bDMARDs from 3111 patients were included, of which 1136 TCs (27%) were initiated as monotherapy. bDMARD monotherapy was preferentially prescribed to older, co-morbid patients with longer disease duration, lower BMI, more active disease and more previous bDMARDs. After adjusting for potential confounding factors, drug retention was significantly lower in monotherapy hazard ratio 1.15 (95% CI: 1.03, 1.30). Other factors such as type of bDMARD and calendar year of prescription were associated with a stronger effect on drug retention. Response to treatment in terms of DAS28 evolution was also slightly but significantly less favourable in monotherapy (P = 0.04).
Our data suggest that bDMARD monotherapy is prescribed to more complex cases and is significantly less effective than bDMARD therapy in combination with sDMARDs, but to an extent that is clinically only marginally relevant.
Three randomized controlled trials in early severe systemic sclerosis demonstrated that autologous hematopoietic stem cell transplantation was superior to standard cyclophosphamide therapy. This ...European Society for Blood and Marrow Transplantation multi-center prospective non-interventional study was designed to further decipher efficacy and safety of this procedure for severe systemic sclerosis patients in real-life practice and to search for prognostic factors. All consecutive adult systemic sclerosis patients undergoing a first autologous hematopoietic stem cell transplantation between December 2012 and February 2016 were prospectively included in the study. Primary endpoint was progression free survival. Secondary endpoints were overall survival, non-relapse mortality, response and incidence of progression. Eighty systemic sclerosis patients were included. Median follow-up duration was 24 (6-57) months after stem cell transplantation using cyclophosphamide plus antithymocyte globulins conditioning for all, with CD34+ selection in 35 patients. At 2 years, progression free survival was 81.8%, overall survival was 90%, response was 88.7% and incidence of progression was 11.9%. The 100 days non-relapse mortality was 6.25% (n=5) with four deaths from cardiac event, including three due to cyclophosphamide toxicity. Modified Rodnan skin score and forced vital capacity improved with time (p< 0.001). By multivariate analysis, baseline skin score >24 and older age at transplant were associated with lower progression free survival (Hazard ration 3.32) and 1.77, respectively). CD34+-selection was associated with better response (Hazard ration: 0.46). This study confirms the efficacy of autologous stem cell transplantation in real-life practice for severe systemic sclerosis using non myeloablative conditioning. Careful cardio-pulmonary assessment to identify organ involvement at patient referral, reduced cyclophosphamide doses and CD34+ selection may improve outcomes. The study was registered at ClinicalTrials.gov: NCT02516124.
These updated EBMT guidelines review the clinical evidence, registry activity and mechanisms of action of haematopoietic stem cell transplantation (HSCT) in multiple sclerosis (MS) and other ...immune-mediated neurological diseases and provide recommendations for patient selection, transplant technique, follow-up and future development. The major focus is on autologous HSCT (aHSCT), used in MS for over two decades and currently the fastest growing indication for this treatment in Europe, with increasing evidence to support its use in highly active relapsing remitting MS failing to respond to disease modifying therapies. aHSCT may have a potential role in the treatment of the progressive forms of MS with a significant inflammatory component and other immune-mediated neurological diseases, including chronic inflammatory demyelinating polyneuropathy, neuromyelitis optica, myasthenia gravis and stiff person syndrome. Allogeneic HSCT should only be considered where potential risks are justified. Compared with other immunomodulatory treatments, HSCT is associated with greater short-term risks and requires close interspeciality collaboration between transplant physicians and neurologists with a special interest in these neurological conditions before, during and after treatment in accredited HSCT centres. Other experimental cell therapies are developmental for these diseases and patients should only be treated on clinical trials.
Recently, serious infections related to the use of tofacitinib (TOF) for treatment of rheumatoid arthritis (RA) have raised considerable interest. This study aimed to compare the risk for serious ...infections in patients with RA upon receiving TOF versus biologic disease-modifying antirheumatic drugs (bDMARDs) by age at treatment initiation. We identified adult RA patients exposed to TOF or bDMARDs using data collected by the Swiss registry for inflammatory rheumatic diseases (SCQM) from 2015 to 2018. The event of interest was the first non-fatal serious infection (SI) during drug exposure. Missing or incomplete SI dates were imputed as either the lower (left) or upper (right) limit of the known occurrence interval. The ratio of SI hazards (HR) of TOF versus bDMARDs was estimated as a function of age using covariate-adjusted Cox regression applied to each type of imputed time-to-SI. A total of 1687 patients provided time at risk for a first SI during study participation and drug exposure for 2238 different treatment courses, 345 for TOF and 1893 for bDMARDs. We identified 44 (left imputation) or 43 (right imputation), respectively, first SIs (12/12 on TOF versus 32/31 on bDMARDs). Left and right imputation produced similar results. For patients aged ≥ 69 years, the treatment HR started to be increased (lower limit of 95% confidence intervals (LLCIs) > 1). By the age of 76, the difference between TOF and bDMARDs started to be clinically relevant (LLCIs > 1.25). For patients aged < 65 years, the data were insufficient to draw conclusions. Our results suggest that we should expect an increased risk for SIs in older patients treated with TOF compared to bDMARDs supporting a cautious use of TOF in these patients.
We examined the validity of 5 successful aging (SA) operationalizations that assessed different facets of the SA construct (cognitive and physical health and disability; well-being; social ...engagement).
A total of 2,478 participants (mean age = 82.5 years, standard deviation SD = 3.47) were studied. We used confirmatory factor analysis to investigate the relationships between facets and to determine the convergent validity as well as short-term (1.5 years) and long-term (4.5 years) predictive validity of the 5 SA operationalizations for measures of quality of life (QoL) and objective health outcomes.
A general SA operationalization that included all SA facets but also allowed differences between them showed the best model fit and construct validity. A biomedical operationalization of SA that excluded either the well-being or the social engagement facet showed lower convergent and predictive validity for subjective measures (e.g., QoL) but higher associations with objective measures (e.g., health). A purely psychosocial SA operationalization that excluded the physiological facet did not allow good prediction of objective health outcomes.
Our results suggest that a well-balanced SA operationalization should include measures assessing health, disability, well-being, and social engagement.
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