Andreas Woerner

Amyloid-like Aggregates Sequester Numerous Metastable Proteins with Essential Cellular Functions

Protein aggregation is linked with neurodegeneration and numerous other diseases by mechanisms that are not well understood. Here, we have analyzed the gain-of-function toxicity of artificial β sheet proteins that were designed to form amyloid-like fibrils. Using quantitative proteomics, we found that the toxicity of these proteins in human cells correlates with the capacity of their aggregates to promote aberrant protein interactions and to deregulate the cytosolic stress response. The endogenous proteins that are sequestered by the aggregates share distinct physicochemical properties: They are relatively large in size and significantly enriched in predicted unstructured regions, features that are strongly linked with multifunctionality. Many of the interacting proteins occupy essential hub positions in cellular protein networks, with key roles in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. We suggest that amyloidogenic aggregation targets a metastable subproteome, thereby causing multifactorial toxicity and, eventually, the collapse of essential cellular functions.

The cellular prion protein mediates neurotoxic signalling of β‐sheet‐rich conformers independent of prion replication

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimers disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease‐associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrPSc), a β‐sheet‐rich isoform of the cellular PrP (PrPC), are dependent on neuronal expression of PrPC. In this study, we demonstrate that PrPC has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β‐sheet‐rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β‐peptide, (iii) yeast prion proteins or (iv) designed β‐peptides. Toxic signalling via PrPC requires the intrinsically disordered N‐terminal domain (N‐PrP) and the GPI anchor of PrP. We found that the N‐terminal domain is important for mediating the interaction of PrPC with β‐conformers. Interestingly, a secreted version of N‐PrP associated with β‐conformers and antagonized their toxic signalling via PrPC. Moreover, PrPC‐mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer‐specific antibody. Our study indicates that PrPC can mediate toxic signalling of various β‐sheet‐rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.

Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA

Location, location, location Aggregates of certain disease-associated proteins are involved in neurodegeneration. Woerner et al. now show that the exact location of these aggregates in the cell may be the key to their pathology (see the Perspective by Da Cruz and Cleveland). An artificial aggregate-prone protein caused problems when expressed in the cytoplasm but not when expressed in the nucleus. Cytoplasmic aggregates interfered with nucleocytoplasmic import and export. Perhaps if we can shunt pathological aggregates to the nucleus in the future, we will be able to ameliorate some forms of degenerative disease. Science, this issue p. 173; see also p. 125 Protein aggregates in the cytoplasm soak up accessory factors needed for transport of other proteins and RNA across the nuclear envelope. [Also see Perspective by Da Cruz and Cleveland] Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial β-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein–43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases.

His75―Asp97 Cluster in Green Proteorhodopsin

The proteorhodopsin (PR) family found in bacteria near the oceans surface consists of hundreds of PR variants color-tuned to their environment. PR contains a highly conserved single histidine at position 75, which is not found in most other retinal proteins. Using (13)C and (15)N MAS NMR, we were able to prove for green PR that His75 forms a pH-dependent H-bond with the primary proton acceptor Asp97, which explains its unusually high pK(a). The functional role of His75 has been studied using site-directed mutagenesis and time-resolved optical spectroscopy: Ultrafast vis-pump/vis-probe experiments on PR(H75N) showed that the primary reaction dynamics is retained, while flash photolysis experiments revealed an accelerated photocycle. Our data show the formation of a pH-dependent His-Asp cluster which might be typical for eubacterial retinal proteins. Despite its stabilizing function, His75 was found to slow the photocycle in wild-type PR. This means that PR was not optimized by evolution for fast proton transfer, which raises questions about its true function in vivo.

Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Proteorhodopsin (PR), a light-driven proton pump from marine proteobacteria, exhibits photocycle characteristics similar to bacteriorhodopsin (BR) at neutral pH, including an M-like photointermediate. However, at acidic pH, spectroscopic evidence for an M-like species was absent, and the vectoriality of proton pumping was inverted. To gain further insight into this unusual property, we examined the voltage dependence of stationary and laser flash-induced photocurrents of PR under different pH conditions upon expression in Xenopus oocytes. The current-voltage curves were linear under all conditions tested, and photocurrent reversal potentials distinctly depended on the pH gradient. PR mutants D97N and D97T exhibited transient and stationary inward currents already at neutral pH, showing that neutralization of the proton acceptor abolishes forward pumping and permits only inward proton transport. Mutation E108G, which disrupts the donor site for Schiff base (SB) reprotonation, resulted in largely reduced photocurrents, which could be strongly stimulated by azide, similar to previous observations on BR mutant D96G. When PR and BR photocurrents in response to blue or green laser flashes during or after continuous illumination were compared, direct electrical evidence for the occurrence of an M-like intermediate at neutral pH could only be obtained when reprotonation of the SB was slowed down by PR mutation E108G. For PR at acidic pH, laser flashes only produced inwardly directed photocurrents, independent from background illumination, thus precluding electrical identification of an M-like species. However, when visible absorption spectroscopy was carried out at low temperatures, occurrence of an M-like species was robustly observed at low pH. This indicates that SB deprotonation and reprotonation occur during the PR photocycle also at low pH. Our results corroborate the conclusion that in PR, the direction of proton pumping can be switched by changes in pH and membrane potential, with the protonation state of Asp-97 being the key determinant for selecting between transport modes.

Biological treatment in systemic juvenile idiopathic arthritis: achievement of inactive disease or clinical remission on a first, second or third biological agent.

Objectives To analyse the effect of biological agents (BAs) in terms of achieving inactive disease (ID) or clinical remission (CR) in patients with systemic juvenile idiopathic arthritis (SJIA), to describe effects of switching or discontinuing a BA and to assess the proportion of patients able to maintain ID or CR off steroids and after withdrawing BA therapy. Methods Retrospective study in a French paediatric rheumatology reference centre using the CEMARA (CEntre des MAladies RAres) register. Results Seventy-seven patients were included with a cumulative follow-up of 245.5 patient-years (median 1.1, range 0.5–8.0). On a first BA, ID was achieved in 37 patients, including 1 patient out of 12 patients on etanercept, 26 patients out of 51 on anakinra and 7 out of 10 on canakinumab. One patient on abatacept and two patients on tocilizumab also achieved ID. Switching of BA was common. The switch to a second (n=34), third (n=18) or fourth (n=4) BA resulted in ID in a further 13 patients, either on canakinumab (n=6) or tocilizumab (n=7). At last follow-up, 40 patients were in CR (27 patients off steroids, 5 patients having never received steroid treatment), either on (n=29) or off (n=11) BA. Conclusions In this series of patients with SJIA, interleukin-1 inhibitors were associated with a higher proportion of ID than tumour necrosis factor inhibitors when used as first BA. Switching allowed some patients to achieve ID when treated with canakinumab or tocilizumab. CR was eventually achieved in more than half of the patients.

Immune response to influenza vaccination in children treated with methotrexate or/and tumor necrosis factor-α inhibitors

In children treated with immunosuppressive medication such as methotrexate and tumor necrosis factor-alpha (TNF-α) inhibitors, additional immunizations are recommended because of increased susceptibility to infections. However, it is unclear if adequate antibody response to vaccinations can be established in children receiving methotrexate and/or TNF-α inhibitors. In a prospective open label study, we assessed seroprotection and seroconversion following influenza vaccination during 2 seasons (6 strains) in 36 children with autoimmune disease treated either with methotrexate (n=18), TNF-α inhibitors (n=10) or both (n=8) and a control group of 16 immunocompetent children. Influenza antibody titers were determined by hemagglutinin inhibition assay, before and 4-8 weeks after vaccination. Post-vaccination seroprotection (defined as a titer ³1:40) did not significantly differ between immunosuppressed and immunocompetent subjects. Seroconversion, defined as the change from a nonprotective (

Complications of systemic juvenile idiopathic arthritis: risk factors and management recommendations

Systemic juvenile idiopathic arthritis (SJIA) is an inflammatory condition characterized by fever, lymphadenopathy, arthritis, rash and serositis. Systemic inflammation has been associated with dysregulation of the innate immune system, suggesting that SJIA is an autoinflammatory disorder. IL-1 and IL-6 play a major role in the pathogenesis of SJIA, and treatment with IL-1 and IL-6 inhibitors has shown to be highly effective. However, complications of SJIA, including macrophage activation syndrome, limitations in functional outcome by arthritis and long-term damage from chronic inflammation, continue to be a major issue in SJIA patients’ care. Translational research leading to a profound understanding of the cytokine crosstalk in SJIA and the identification of risk factors for SJIA complications will help to improve long-term outcome.

Effect of Biologic Therapy on Clinical and Laboratory Features of Macrophage Activation Syndrome Associated With Systemic Juvenile Idiopathic Arthritis

To assess performance of the 2016 macrophage activation syndrome (MAS) classification criteria for patients with systemic juvenile idiopathic arthritis (JIA) who develop MAS while treated with biologic medications.

Neonatological and pulmonological management of bilateral pulmonary sequestration in a neonate

BackgroundBronchopulmonary sequestration is a lung malformation characterized by nonfunctioning lung tissue without primary communication with the tracheobronchial tree. Intrauterine complications such as mediastinal shift, pleural effusion or fetal hydrothorax can be present. We present the case of a newborn with bilateral intralobar pulmonary sequestration.MethodsPrenatal ultrasonography in a primigravida at 20 weeks of gestation revealed echogenic masses in the right fetal hemithorax with mediastinal shift towards the left side. Serial ultrasound confirmed persistence of the lesion with otherwise appropriate fetal development. Delivery was uneventful and physical examination revealed an isolated intermittent tachypnea. Chest CT scan and CT angiography showed a bilateral intrathoracic lesion with arterial supply from the aorta. Baby lung function testing suggested possible multiple functional compartments.ResultsRight and left thoracotomy was performed at the age of 7 months. A bilateral intralobar sequestration with vascularisation from the aorta was resected. Pathological and histological examination of the resected tissue confirmed the surgical diagnosis. At the age of 24 months, the child was doing well without pulmonary complications.ConclusionsBilateral pulmonary sequestration requires intensive prenatal and postnatal surveillance. Though given the fact of a bilateral pulmonary sequestration, postnatal outcome showed similar favourable characteristics to an unilateral presentation. Baby lung function testing could provide additional information for optimal postnatal management and timing of surgical intervention.