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Abstract Nucleoporins rich in phenylalanine/glycine (FG) residues form the permeability barrier within the nuclear pore complex and are implicated in several pathological cellular processes, including oncogenic fusion condensates. The self-association of FG-repeat proteins and interactions between FG-repeats play a critical role in these activities by forming hydrogel-like structures. Here we show that mutation of specific FG repeats of Nup98 can strongly decrease the protein’s self-association capabilities. We further present a cryo-electron microscopy structure of a Nup98 peptide fibril with higher stability per residue compared with previous Nup98 fibril structures. The high-resolution structure reveals zipper-like hydrophobic patches which contain a GLFG motif and are less compatible for binding to nuclear transport receptors. The identified distinct molecular properties of different regions of the nucleoporin may contribute to spatial variations in the self-association of FG-repeats, potentially influencing transport processes through the nuclear pore.

Abstract Abnormal trinucleotide repeat expansions alter protein conformation causing malfunction and contribute to a significant number of incurable human diseases. Scarce structural insights available on disease-related homorepeat expansions hinder the design of effective therapeutics. Here, we present the dynamic structure of human PHOX2B C-terminal fragment, which contains the longest polyalanine segment known in mammals. The major α-helical conformation of the polyalanine tract is solely extended by polyalanine expansions in PHOX2B, which are responsible for most congenital central hypoventilation syndrome cases. However, polyalanine expansions in PHOX2B additionally promote nascent homorepeat conformations that trigger length-dependent phase transitions into solid condensates that capture wild-type PHOX2B. Remarkably, HSP70 and HSP90 chaperones specifically seize PHOX2B alternative conformations preventing phase transitions. The precise observation of emerging polymorphs in expanded PHOX2B postulates unbalanced phase transitions as distinct pathophysiological mechanisms in homorepeat expansion diseases, paving the way towards the search of therapeutics modulating biomolecular condensates in central hypoventilation syndrome.

Abstract Alzheimer’s disease begins with mild memory loss and slowly destroys memory and thinking. Cognitive impairment in Alzheimer’s disease has been associated with the localization of the microtubule-associated protein Tau at the postsynapse. However, the correlation between Tau at the postsynapse and synaptic dysfunction remains unclear. Here, we show that Tau arrests liquid-like droplets formed by the four postsynaptic density proteins PSD-95, GKAP, Shank, Homer in solution, as well as NMDA (N-methyl-D-aspartate)-receptor-associated protein clusters on synthetic membranes. Tau-mediated condensate/cluster arrest critically depends on the binding of multiple interaction motifs of Tau to a canonical GMP-binding pocket in the guanylate kinase domain of PSD-95. We further reveal that competitive binding of a high-affinity phosphorylated peptide to PSD-95 rescues the diffusional dynamics of an NMDA truncated construct, which contains the last five amino acids of the NMDA receptor subunit NR2B fused to the C-terminus of the tetrameric GCN4 coiled-coil domain, in postsynaptic density-like condensates/clusters. Taken together, our findings propose a molecular mechanism where Tau modulates the dynamic properties of the postsynaptic density.

Abstract Eukaryotic gene regulation and pre-mRNA transcription depend on the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II. Due to its highly repetitive, intrinsically disordered sequence, the CTD enables clustering and phase separation of Pol II. The molecular interactions that drive CTD phase separation and Pol II clustering are unclear. Here, we show that multivalent interactions involving tyrosine impart temperature- and concentration-dependent self-coacervation of the CTD. NMR spectroscopy, molecular ensemble calculations and all-atom molecular dynamics simulations demonstrate the presence of diverse tyrosine-engaging interactions, including tyrosine-proline contacts, in condensed states of human CTD and other low-complexity proteins. We further show that the network of multivalent interactions involving tyrosine is responsible for the co-recruitment of the human Mediator complex and CTD during phase separation. Our work advances the understanding of the driving forces of CTD phase separation and thus provides the basis to better understand CTD-mediated Pol II clustering in eukaryotic gene transcription.

Abstract Pathogenic aggregation of the protein tau is a hallmark of Alzheimer’s disease and several other tauopathies. Tauopathies are characterized by the deposition of specific tau isoforms as disease-related tau filament structures. The molecular processes that determine isoform-specific deposition of tau are however enigmatic. Here we show that acetylation of tau discriminates its isoform-specific aggregation. We reveal that acetylation strongly attenuates aggregation of four-repeat tau protein, but promotes amyloid formation of three-repeat tau. We further identify acetylation of lysine 298 as a hot spot for isoform-specific tau aggregation. Solid-state NMR spectroscopy demonstrates that amyloid fibrils formed by unmodified and acetylated three-repeat tau differ in structure indicating that site-specific acetylation modulates tau structure. The results implicate acetylation as a critical regulator that guides the selective aggregation of three-repeat tau and the development of tau isoform-specific neurodegenerative diseases.

Abstract Preventing tau aggregation is a potential therapeutic strategy in Alzheimer’s disease and other tauopathies. Recently, liquid–liquid phase separation has been found to facilitate the formation of pathogenic tau conformations and fibrillar aggregates, although many aspects of the conformational transitions of tau during the phase transition process remain unknown. Here, we demonstrate that the tau aggregation inhibitor methylene blue promotes tau liquid–liquid phase separation and accelerates the liquid-to-gel transition of tau droplets independent of the redox activity of methylene blue. We further show that methylene blue inhibits the conversion of tau droplets into fibrils and reduces the cytotoxicity of tau aggregates. Although gelation slows down the mobility of tau and tubulin, it does not impair microtubule assembly within tau droplets. These findings suggest that methylene blue inhibits tau amyloid fibrillization and accelerates tau droplet gelation via distinct mechanisms, thus providing insights into the activity of tau aggregation inhibitors in the context of phase transition.

In this work, the authors identify Genipin as a small iridoid able to prevent alpha-synuclein aggregation and toxicity by affecting endocytosis, metabolism and lipid storage.

Quaternary structure of α-synuclein fibrils characterized from human patients with Parkinson’s disease and Multiple System Atrophy modulates seeding in mouse primary oligodendroglial cultures.

Alzheimer’s disease is characterized by the accumulation of aggregated tau protein. Here the authors find that Hsp chaperones, which normally protect cell homeostasis, can assemble with co-chaperones in a “multichaperone machinery” to target tau aggregation.

Abstract Abnormal deposition of α-synuclein aggregates in Lewy bodies and Lewy neurites is the hallmark lesion in Parkinson’s disease (PD). These aggregates, thought to be the culprit of disease pathogenesis, spread throughout the brain as the disease progresses. Agents that inhibit α-synuclein aggregation and/or spread of aggregates would thus be candidate disease-modifying drugs. Here, we found that Chicago sky blue 6B (CSB) may be such a drug, showing that it inhibits α-synuclein aggregation and cell-to-cell propagation in both in vitro and in vivo models of synucleinopathy. CSB inhibited the fibrillation of α-synuclein in a concentration-dependent manner through direct binding to the N-terminus of α-synuclein. Furthermore, both seeded polymerization and cell-to-cell propagation of α-synuclein were inhibited by CSB treatment. Notably, CSB alleviated behavioral deficits and neuropathological features, such as phospho-α-synuclein and astrogliosis, in A53T α-synuclein transgenic mice. These results indicate that CSB directly binds α-synuclein and inhibits its aggregation, thereby blocking α-synuclein cell-to-cell propagation.

Abstract Background Alzheimer’s disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. Methods We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. Results While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive β-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. Conclusions ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.

The authors present a method for the conversion of full-length tau protein into seeding-competent amyloid fibrils without heparin or other negatively charged co-factors, which could be useful for studying the effects of post-translational modifications on Tau aggregation as well as to identify potential inhibitors of tau aggregation. Biochemical experiments and solid-state NMR spectroscopy measurements show that these co-factor-free tau fibrils have similar properties as amyloid fibrils isolated from brain tissue but differ from those of commonly used heparin-induced tau fibrils.

Abstract With the advent of the genetic era in Parkinson’s disease (PD) research in 1997, α-synuclein was identified as an important player in a complex neurodegenerative disease that affects >10 million people worldwide. PD has been estimated to have an economic impact of $51.9 billion in the US alone. Since the initial association with PD, hundreds of researchers have contributed to elucidating the functions of α-synuclein in normal and pathological states, and these remain critical areas for continued research. With this position paper the authors strive to achieve two goals: first, to succinctly summarize the critical features that define α-synuclein’s varied roles, as they are known today; and second, to identify the most pressing knowledge gaps and delineate a multipronged strategy for future research with the goal of enabling therapies to stop or slow disease progression in PD.

The most frequent cause of familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are hexanucleotide repeat expansions in the non-coding region of the C9ORF72 gene that are translated into five dipeptide repeat (DPR) proteins. Here, the authors show that proline/arginine (PR) DPRs inhibit the prolyl isomerase PPIA and reveal the molecular mechanism of the impaired protein folding activity of PPIA by performing NMR measurements and determining a PR DPR bound PPIA crystal structure.

The SARS-CoV-2 viral genome is encapsulated by the nucleocapsid protein (NSARS-CoV-2) that is essential for viral replication. Here, the authors show that RNA induces liquid-liquid phase separation of NSARS-CoV-2 and how NSARS-CoV-2 phosphorylation modulates RNA-binding and phase separation and that these RNA/NSARS-CoV-2-droplets recruit and concentrate the SARS-CoV-2 RNA-dependent RNA polymerase complex in vitro, which would enable high initiation and elongation rates during viral transcription.

Cyclophilin A (CypA) is a peptidylprolyl isomerase that also has chaperone activity and interacts with the intrinsically disordered protein α-Synuclein (aSyn). Here, the authors combine NMR measurements and biochemical experiments to characterise the interplay between the catalysis of proline isomerization and molecular chaperone activity of CypA and find that both activities have opposing effects on aSyn and further show that the that cis/trans isomerization outpowers the holding activity of CypA.

α-Syn in CSF is a biomarker of neurodegenerative diseases; however, the detection of clinically relevant species is difficult. Here, the authors create a nanobody biosensor that reveals the presence of α-Syn in cells, which allow the detection of transmittable forms of α-Syn present in human CSF.

Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are characterized by the pathological accumulation of α-synuclein. Here the authors employ fluorescent probes, electron microscopy and NMR spectroscopy to study the properties of α-synuclein aggregates that were amplified from patient brain extracts and observe a greater structural diversity among PD patients compared to MSA patients.

α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson’s disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked hαSynTP or hαSynΔ119 (h stands for “human”) into the murine αSyn locus. hαSynTP is a structure-based mutant with triple alanine to proline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster. hαSynΔ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of hαSynTP or hαSynΔ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that hαSynTP and hαSynΔ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSynTP and hαSynΔ119 mice, despite the lower levels of hαSynTP and hαSynΔ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSynΔ119 mice, while a compulsive behavior was exclusively detected in hαSynTP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches.

Processing bodies (P-bodies) are non-membrane-bound protein/RNA granules in the cytosol. Here the authors combine bioinformatics, NMR and cell based assays and find that lysine is enriched in the disordered regions of P-body-associated proteins and show that lysine-rich polypeptides form highly dynamic lysine/RNA-coacervates and lysine acetylation reverses liquid-liquid phase separation.

Tauopathies are neurodegenerative disorders with increasing incidence and still without cure. The extensive time required for development and approval of novel therapeutics highlights the need for testing and repurposing known safe molecules. Since doxycycline impacts α-synuclein aggregation and toxicity, herein we tested its effect on tau. We found that doxycycline reduces amyloid aggregation of the 2N4R and K18 isoforms of tau protein in a dose-dependent manner. Furthermore, in a cell free system doxycycline also prevents tau seeding and in cell culture reduces toxicity of tau aggregates. Overall, our results expand the spectrum of action of doxycycline against aggregation-prone proteins, opening novel perspectives for its repurposing as a disease-modifying drug for tauopathies.

Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.

The chaperone Hsp90 plays a key role in maintaining cellular homeostasis. Here the authors provide structural insights into substrate recognition and the pro-folding mechanism of Hsp90/co-chaperone complexes by studying the complex of Hsp90 with its co-chaperone FKBP51 and the substrate Tau bound Hsp90/FKBP51 ternary complex using a NMR based integrative approach.

Abstract Alzheimer's disease is a devastating neurodegenerative disease eventually leading to dementia. An effective treatment does not yet exist. Here we show that oral application of the compound anle138b restores hippocampal synaptic and transcriptional plasticity as well as spatial memory in a mouse model for Alzheimer's disease, when given orally before or after the onset of pathology. At the mechanistic level, we provide evidence that anle138b blocks the activity of conducting Aβ pores without changing the membrane embedded Aβ‐oligomer structure. In conclusion, our data suggest that anle138b is a novel and promising compound to treat AD‐related pathology that should be investigated further.

The microtubule associated protein Tau also interacts with filamentous actin. Here the authors combine biophysical experiments and NMR studies to characterize the structural changes that occur in Tau upon binding to filamentous actin and show that phosphorylation of serine 262 attenuates actin binding of Tau.

Delivery of neurotrophins and neuropeptides via long-range trafficking of dense core vesicles (DCVs) from the cell soma to nerve terminals is essential for synapse modulation and circuit function. But the mechanism by which transiting DCVs are captured at specific sites is unknown. Here, we discovered that Synaptotagmin-4 (Syt4) regulates the capture and spatial distribution of DCVs in hippocampal neurons. We found that DCVs are highly mobile and undergo long-range translocation but switch directions only at the distal ends of axons, revealing a circular trafficking pattern. Phosphorylation of serine 135 of Syt4 by JNK steers DCV trafficking by destabilizing Syt4-Kif1A interaction, leading to a transition from microtubule-dependent DCV trafficking to capture at en passant presynaptic boutons by actin. Furthermore, neuronal activity increased DCV capture via JNK-dependent phosphorylation of the S135 site of Syt4. Our data reveal a mechanism that ensures rapid, site-specific delivery of DCVs to synapses.

Tau forms aggregates in the brains of Alzheimer patients. Here, the authors identify conditions, where the microtubule-binding repeats of Tau undergo a phosphorylation-dependent liquid–liquid phase separation, leading to molecular crowding in the formed Tau liquid droplets and characterize them by NMR and other biophysical methods.

The outer mitochondrial membrane translocator protein (TSPO) mediates several mitochondrial functions and binds cholesterol with a high affinity. Here the authors use solid-state NMR to show that cholesterol binding to TSPO results in allosteric changes that modulate TSPO oligomerization.

Protein aggregation plays a crucial role in several neurodegenerative diseases. Here the authors demonstrate that phosphorylation of β-amyloid aggregates—the pathological hallmark of Alzheimer's disease—can change the molecular properties of aggregates, suggesting how phosphorylation contributes to disease progression.

α-Synuclein (α-Syn) is intimately linked to the etiology of Parkinson's Disease, as mutations and even subtle increases in gene dosage result in early onset of the disease. However, how this protein causes neuronal dysfunction and neurodegeneration is incompletely understood. We thus examined a comprehensive range of physiological parameters in cultured rat primary neurons overexpressing α-Syn at levels causing a slowly progressive neurodegeneration. In contradiction to earlier reports from non-neuronal assay systems we demonstrate that α-Syn does not interfere with essential ion handling capacities, mitochondrial capability of ATP production or basic electro-physiological properties like resting membrane potential or the general ability to generate action potentials. α-Syn also does not activate canonical stress kinase Signaling converging on SAPK/Jun, p38 MAPK or Erk kinases. Causative for α-Syn-induced neurodegeneration are mitochondrial thiol oxidation and activation of caspases downstream of mitochondrial outer membrane permeabilization, leading to apoptosis-like cell death execution with some unusual aspects. We also aimed to elucidate neuroprotective strategies counteracting the pathophysiological processes caused by α-Syn. Neurotrophic factors, calpain inhibition and increased lysosomal protease capacity showed no protective effects against α-Syn overexpression. In contrast, the major watchdog of outer mitochondrial membrane integrity, Bcl-Xl, was capable of almost completely preventing neuron death, but did not prevent mitochondrial thiol oxidation. Importantly, independent from the quite mono-causal induction of neurotoxicity, α-Syn causes diminished excitability of neurons by external stimuli and robust impairments in endogenous neuronal network activity by decreasing the frequency of action potentials generated without external stimulation. This latter finding suggests that α-Syn can induce neuronal dysfunction independent from its induction of neurotoxicity and might serve as an explanation for functional deficits that precede neuronal cell loss in synucleopathies like Parkinson's disease or dementia with Lewy bodies.

The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.

[This corrects the article DOI: 10.1371/journal.pbio.2000374.].

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.

Alpha-synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 – the Drosophila ortholog of Rab8a – ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS–Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.

The stability and dynamics of cytoskeleton in brain nerve cells are regulated by microtubule associated proteins (MAPs), tau and MAP2. Both proteins are intrinsically disordered and involved in multiple molecular interactions important for normal physiology and pathology of chronic neurodegenerative diseases. Nuclear magnetic resonance and cryo-electron microscopy recently revealed propensities of MAPs to form transient local structures and long-range contacts in the free state, and conformations adopted in complexes with microtubules and filamentous actin, as well as in pathological aggregates. In this paper, we compare the longest, 441-residue brain isoform of tau (tau40), and a 467-residue isoform of MAP2, known as MAP2c. For both molecules, we present transient structural motifs revealed by conformational analysis of experimental data obtained for free soluble forms of the proteins. We show that many of the short sequence motifs that exhibit transient structural features are linked to functional properties, manifested by specific interactions. The transient structural motifs can be therefore classified as molecular recognition elements of tau40 and MAP2c. Their interactions are further regulated by post-translational modifications, in particular phosphorylation. The structure-function analysis also explains differences between biological activities of tau40 and MAP2c.

Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.

Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.

As essential components of the molecular machine assembling heterochromatin in eukaryotes, HP1 (Heterochromatin Protein 1) proteins are key regulators of genome function. While several high-resolution structures of the two globular regions of HP1, chromo and chromoshadow domains, in their free form or in complex with recognition-motif peptides are available, less is known about the conformational behavior of the full-length protein. Here, we used NMR spectroscopy in combination with small angle X-ray scattering and dynamic light scattering to characterize the dynamic and structural properties of full-length human HP1β (hHP1β) in solution. We show that the hinge region is highly flexible and enables a largely unrestricted spatial search by the two globular domains for their binding partners. In addition, the binding pockets within the chromo and chromoshadow domains experience internal dynamics that can be useful for the versatile recognition of different binding partners. In particular, we provide evidence for the presence of a distinct structural propensity in free hHP1β that prepares a binding-competent interface for the formation of the intermolecular β-sheet with methylated histone H3. The structural plasticity of hHP1β supports its ability to bind and connect a wide variety of binding partners in epigenetic processes.

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Conformational changes are essential for protein-protein and protein-ligand recognition. Here we probed changes in the structure of the protein ubiquitin at low temperatures in supercooled water using NMR spectroscopy. We demonstrate that ubiquitin is well folded down to 263 K, although slight rearrangements in the hydrophobic core occur. However, amide proton chemical shifts show non-linear temperature dependence in supercooled solution and backbone hydrogen bonds become weaker in the region that is most prone to cold-denaturation. Our data suggest that the weakening of the hydrogen bonds in the β-sheet of ubiquitin might be one of the first events that occur during cold-denaturation of ubiquitin. Interestingly, the same region is strongly involved in ubiquitin-protein complexes suggesting that this part of ubiquitin more easily adjusts to conformational changes required for complex formation.

Parkinson's disease (PD) is linked to the formation of insoluble fibrillar aggregates of the presynaptic protein α-Synuclein (αS) in neurons. The appearance of such aggregates coincides with severe motor deficits in human patients. These deficits are often preceded by non-motor symptoms such as sleep-related problems in the patients. PD-like motor deficits can be recapitulated in model organisms such as Drosophila melanogaster when αS is pan-neurally expressed. Interestingly, both these deficits are more severe when αS mutants with reduced aggregation properties are expressed in flies. This indicates that that αS aggregation is not the primary cause of the PD-like motor symptoms. Here we describe a model for PD in Drosophila which utilizes the targeted expression of αS mutants in a subset of dopadecarboxylase expressing serotonergic and dopaminergic (DA) neurons. Our results show that targeted expression of pre-fibrillar αS mutants not only recapitulates PD-like motor symptoms but also the preceding non-motor symptoms such as an abnormal sleep-like behavior, altered locomotor activity and abnormal circadian periodicity. Further, the results suggest that the observed non-motor symptoms in flies are caused by an early impairment of neuronal functions rather than by the loss of neurons due to cell death.

Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.

Alzheimer disease is characterized by abnormal protein deposits in the brain, such as extracellular amyloid plaques and intracellular neurofibrillary tangles. The tangles are made of a protein called tau comprising 441 residues in its longest isoform. Tau belongs to the class of natively unfolded proteins, binds to and stabilizes microtubules, and partially folds into an ordered beta-structure during aggregation to Alzheimer paired helical filaments (PHFs). Here we show that it is possible to overcome the size limitations that have traditionally hampered detailed nuclear magnetic resonance (NMR) spectroscopy studies of such large nonglobular proteins. This is achieved using optimal NMR pulse sequences and matching of chemical shifts from smaller segments in a divide and conquer strategy. The methodology reveals that 441-residue tau is highly dynamic in solution with a distinct domain character and an intricate network of transient long-range contacts important for pathogenic aggregation. Moreover, the single-residue view provided by the NMR analysis reveals unique insights into the interaction of tau with microtubules. Our results establish that NMR spectroscopy can provide detailed insight into the structural polymorphism of very large nonglobular proteins.

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.