Intrinsic disorder in the common N-terminus of human adenovirus 5 E1B-55K and its related E1BN proteins indicated by studies on E1B-93R

AbstractThe E1B transcription unit of human adenovirus encodes at least five different proteins generated by alternative splicing of a common E1B precursor mRNA. E1B-156R, -93R and -84R contain individual carboxy termini but share a common amino terminus. To acquire data on the structure of the amino terminus we performed biophysical analyses on E1B-93R. We show that E1B-93R is mostly unstructured and fulfills the criteria of an intrinsically disordered protein (IDP). The intrinsic disorder in the amino terminus of these proteins is evolutionary conserved in all seven human adenovirus species. As IDPs comprise a rapidly growing family of proteins which, despite their lack of a well defined structure, often fulfill essential regulatory functions, the observations described here might open up a new avenue for the understanding of the regulation and functions of E1B proteins, in particular the multifunctional E1B-55K oncoprotein

SGLT2 inhibitor therapy for transthyretin amyloid cardiomyopathy: early tolerance and clinical response to dapagliflozin.

AIMS Sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve clinical outcomes in heart failure patients with reduced and preserved left ventricular ejection fraction (LVEF), but have not yet been investigated in transthyretin amyloid cardiomyopathy (ATTR-CM). This study aimed to evaluate tolerability, clinical outcomes, and changes in NT-proBNP levels and glomerular filtration rate (GFR) in ATTR-CM patients treated with dapagliflozin. METHODS AND RESULTS Patients with stable, tafamidis-treated ATTR-CM were retrospectively evaluated at the initiation of dapagliflozin and 3 months thereafter. Tafamidis-treated ATTR-CM patients without SGLT2i served as a reference cohort. Overall, SLGT2i therapy was initiated in 34 patients. Seventeen patients with stable disease on tafamidis, who were subsequently started on dapagliflozin, were included in the analysis. Patients selected for SGLT2i presented with signs of advanced disease, evidenced by higher Gillmore disease stage (stage ≥2: 53% vs. 27.5%; P = 0.041), baseline median NT-proBNP [median (IQR) 2668 pg/mL (1314-3451) vs. 1424 (810-2059); P = 0.038] and loop diuretic demand (76.5% vs. 45% of patients; P = 0.044), and lower LVEF (46.6 ± 12.9 vs. 53.7 ± 8.7%; P = 0.019) and GFR (51.8 ± 16.5 vs. 68.5 ± 18.6 mL/min; P = 0.037) compared with the reference cohort. At 3-month follow-up, a numerical decrease in NT-proBNP levels was observed in 13/17 (76.5%) patients in the dapagliflozin (-190 pg/mL, IQR: -1,028-71, P = 0.557) and 27/40 (67.5%) of patients in the control cohort (-115 pg/mL, IQR: -357-105, P = 0.551). Other disease parameters remained stable and no adverse events occurred. CONCLUSIONS In tafamidis-treated ATTR-CM patients, initiation of dapagliflozin was well tolerated. The efficacy of SGLT2i therapy in patients with ATTR-CM needs to be studied in randomized controlled trials

E1B-55K-Mediated Regulation of RNF4 SUMO-Targeted Ubiquitin Ligase Promotes Human Adenovirus Gene Expression

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling

Impact of route of access and stenosis subtype on outcome after transcatheter aortic valve replacement.

INTRODUCTION Previous analyses have reported the outcomes of transcatheter aortic valve replacement (TAVR) for patients with low-flow, low-gradient (LFLG) aortic stenosis (AS), without stratifying according to the route of access. Differences in mortality rates among access routes have been established for high-gradient (HG) patients and hypothesized to be even more pronounced in LFLG AS patients. This study aims to compare the outcomes of patients with LFLG or HG AS following transfemoral (TF) or transapical (TA) TAVR. METHODS A total of 910 patients, who underwent either TF or TA TAVR with a median follow-up of 2.22 (IQR: 1.22-4.03) years, were included in this multicenter cohort study. In total, 146 patients (16.04%) suffered from LFLG AS. The patients with HG and LFLG AS were stratified according to the route of access and compared statistically. RESULTS The operative mortality rates of patients with HG and LFLG were found to be comparable following TF access. The operative mortality rate was significantly increased for patients who underwent TA access [odds ratio (OR): 2.91 (1.54-5.48), p = 0.001] and patients with LFLG AS [OR: 2.27 (1.13-4.56), p = 0.02], which could be corroborated in a propensity score-matched subanalysis. The observed increase in the risk of operative mortality demonstrated an additive effect [OR for TA LFLG: 5.45 (2.35-12.62), p < 0.001]. LFLG patients who underwent TA access had significantly higher operative mortality rates (17.78%) compared with TF LFLG (3.96%, p = 0.016) and TA HG patients (6.36%, p = 0.024). CONCLUSIONS HG patients experienced a twofold increase in operative mortality rates following TA compared with TF access, while LFLG patients had a fivefold increase in operative mortality rates. TA TAVR appears suboptimal for patients with LFLG AS. Prospective studies should be conducted to evaluate alternative options in cases where TF is not possible

Diagnostic performance of cardiac magnetic resonance segmental myocardial strain for detecting microvascular obstruction and late gadolinium enhancement in patients presenting after a ST-elevation myocardial infarction.

Background Microvascular obstruction (MVO) and Late Gadolinium Enhancement (LGE) assessed in cardiac magnetic resonance (CMR) are associated with adverse outcome in patients with ST-elevation myocardial infarction (STEMI). Our aim was to analyze the diagnostic performance of segmental strain for the detection of MVO and LGE. Methods Patients with anterior STEMI, who underwent additional CMR were enrolled in this sub-study of the CARE-AMI trial. Using CMR feature tracking (FT) segmental circumferential peak strain (SCS) was measured and the diagnostic performance of SCS to discriminate MVO and LGE was assessed in a derivation and validation cohort. Results Forty-eight STEMI patients (62 ± 12 years old), 39 (81%) males, who underwent CMR (i.e., mean 3.0 ± 1.5 days) after primary percutaneous coronary intervention (PCI) were included. All patients presented with LGE and in 40 (83%) patients, MVO was additionally present. Segments in all patients were visually classified and 146 (19%) segments showed MVO (i.e., LGE+/MVO+), 308 (40%) segments showed LGE and no MVO (i.e., LGE+/MVO-), and 314 (41%) segments showed no LGE (i.e., LGE-). Diagnostic performance of SCS for detecting MVO segments (i.e., LGE+/MVO+ vs. LGE+/MVO-, and LGE-) showed an AUC = 0.764 and SCS cut-off value was -11.2%, resulting in a sensitivity of 78% and a specificity of 67% with a positive predictive value (PPV) of 30% and a negative predictive value (NPV) of 94% when tested in the validation group. For LGE segments (i.e., LGE+/MVO+ and LGE+/MVO- vs. LGE-) AUC = 0.848 and SCS with a cut-off value of -13.8% yielded to a sensitivity of 76%, specificity of 74%, PPV of 81%, and NPV of 70%. Conclusion Segmental strain in STEMI patients was associated with good diagnostic performance for detection of MVO+ segments and very good diagnostic performance of LGE+ segments. Segmental strain may be useful as a potential contrast-free surrogate marker to improve early risk stratification in patients after primary PCI

Myeloid Sarcoma in the Central Nervous System: Case Report and Review of the Literature

Mijeloidni sarkomi su rijetke pojavnosti uglavnom mijeloblastične leukemije. Njihova pojava u središnjem živčanom sustavu je iznimna, pa je dotična literatura danas ograničena na studije pojedinih slučajeva. Mi opisujemo jo. jedan slučaj, dok je pregled literature poslužio kako bismo ispitali značajke i mogućnosti liječenja mijeloidnog sarkoma središnjega živčanog sustva. U žene stare 61 godinu s akutnom mijeloblastičnom leukemijom (FAB M5) i progresivnom lijevostranom hemiparezom utvrđena je desnostrano parieto-okcipitalno epiduralno oštećenje koje je sličilo meningiomu. Učinjena je djelomična resekcija koja je otkrila mijeloidni sarkom. Pregledom literature utvrdili smo 44 slučaja s dostatnim opisom dijagnoze, liječenja i praćenja do jedne godine. Kod tih bolesnika primijenjeni su različiti načini liječenja. Međutim, bolesnici su imali najbolji postotak jednogodišnjeg preživljenja kad je protokol liječenja uključivao sustavnu kemoterapiju ili zračenje.Myeloid sarcomas are rare manifestations of mainly myeloblastic leukemia. Their occurrence in the central nervous system is exceptional and current literature is limited to case studies. A case is added herewith and a review was performed to investigate clinical characteristics and treatment options of central nervous system myeloid sarcoma. A 61-year-old female with acute myeloblastic leukemia (FAB M5) and progressive left sided hemiparesis showed a right parieto-occipital epidural lesion mimicking meningioma. Partial resection was performed to reveal a myeloid sarcoma. Reviewing the literature we identified 44 cases with sufficient description of the diagnosis, treatment and follow up to one year. In these patients different treatment regimens were applied. However, when systemic chemotherapy or irradiation was included in the treatment regimen, patients showed the best 1-year survival proportion

Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1, following adenovirus infection

ABSTRACT Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection. IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target. </jats:p

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo