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 ß 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.

A protein quality control pathway regulated by linear ubiquitination.

Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.

The cellular prion protein mediates neurotoxic signalling of ß-sheet-rich conformers independent of prion replication.

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's 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 (PrP(Sc)), a ß-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various ß-sheet-rich (ß) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid ß-peptide, (iii) yeast prion proteins or (iv) designed ß-peptides. Toxic signalling via PrP(C) 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 PrP(C) with ß-conformers. Interestingly, a secreted version of N-PrP associated with ß-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various ß-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.

His75-Asp97 cluster in green proteorhodopsin.

The proteorhodopsin (PR) family found in bacteria near the ocean's 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.

Photoisomerization in proteorhodopsin mutant D97N.

The first steps of the photocycle of the D97N mutant of proteorhodopsin (PR) have been investigated by means of ultrafast transient absorption spectroscopy. A comparison with the primary dynamics of native PR and D85N mutant of bacteriorhodopsin is given. Upon photoexcitation of the covalently bound all-trans retinal the excited state decays biexponentially with time constants of 1.4 and 20 ps via a conical intersection, resulting in a 13-cis isomerized retinal. Neither of the two-deactivation channels is significantly preferred. The dynamics is slowed down in comparison with native PR at pH 9 and reaction rates are even lower than for native PR at pH 6, where the primary proton acceptor (Asp97) is protonated. Therefore, the ultrafast isomerization is not only controlled by the charge distribution within the retinal binding pocket. This study shows that in addition to direct electrostatics other effects have to be taken into account to explain the catalytic function of Asp97 in PR on the ultrafast isomerization reaction. This may include sterical interactions and/or bound water molecules within the retinal binding pocket.

Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA.

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 ß-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.

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.

Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR.

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin.