No abstract

The spread of pathological α-synuclein (α-syn) is a crucial event in the progression of Parkinson’s disease (PD). Cell surface receptors such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1) can preferentially bind α-syn in the amyloid over monomeric state to initiate cell-to-cell transmission. However, the molecular mechanism underlying this selective binding is unknown. Here, we perform an array of biophysical experiments and reveal that LAG3 D1 and APLP1 E1 domains commonly use an alkaline surface to bind the acidic C terminus, especially residues 118 to 140, of α-syn. The formation of amyloid fibrils not only can disrupt the intramolecular interactions between the C terminus and the amyloid-forming core of α-syn but can also condense the C terminus on fibril surface, which remarkably increase the binding affinity of α-syn to the receptors. Based on this mechanism, we find that phosphorylation at serine 129 (pS129), a hallmark modification of pathological α-syn, can further enhance the interaction between α-syn fibrils and the receptors. This finding is further confirmed by the higher efficiency of pS129 fibrils in cellular internalization, seeding, and inducing PD-like α-syn pathology in transgenic mice. Our work illuminates the mechanistic understanding on the spread of pathological α-syn and provides structural information for therapeutic targeting on the interaction of α-syn fibrils and receptors as a potential treatment for PD.

Many human neurodegenerative diseases are associated with amyloid fibril formation. Inhibition of amyloid formation is of importance for therapeutics of the related diseases. However, the development of selective potent amyloid inhibitors remains challenging. Here based on the structures of amyloid β (Aβ) fibrils and their amyloid-forming segments, we designed a series of peptide inhibitors using RosettaDesign. We further utilized a chemical scaffold to constrain the designed peptides into β-strand conformation, which significantly improves the potency of the inhibitors against Aβ aggregation and toxicity. Furthermore, we show that by targeting different Aβ segments, the designed peptide inhibitors can selectively recognize different species of Aβ. Our study developed an approach that combines the structure-based rational design with chemical modification for the development of amyloid inhibitors, which could be applied to the development of therapeutics for different amyloid-related diseases.

αB-Crystallin (αBc) is a small heat shock protein that protects cells against abnormal protein aggregation and disease-related degeneration. αBc is also a major structural protein that forms polydisperse multimers that maintain the liquid-like property of the eye lens. However, the relationship and regulation of the two functions have yet to be explored. Here, by combining NMR spectroscopy and multiple biophysical approaches, we found that αBc uses a conserved β4/β8 surface of the central α-crystallin domain to bind α-synuclein and Tau proteins and prevent them from aggregating into pathological amyloids. We noted that this amyloid-binding surface can also bind the C-terminal IPI motif of αBc, which mediates αBc multimerization and weakens its chaperone activity. We further show that disruption of the IPI binding impairs αBc self-multimerization but enhances its chaperone activity. Our work discloses the structural mechanism underlying the regulation of αBc chaperone activity and self-multimerization and sheds light on the different functions of αBc in antagonizing neurodegeneration and maintaining eye lens liquidity.

Vesicle associated membrane protein 2 (VAMP2/synaptobrevin2), a core SNARE protein residing on synaptic vesicles (SVs), forms helix bundles with syntaxin-1 and SNAP25 for the SNARE assembly. Prior to the SNARE assembly, the structure of VAMP2 is unclear. Here, by using in-cell NMR spectroscopy, we describe the dynamic membrane association of VAMP2 SNARE motif in mammalian cells, and the structural change of VAMP2 upon the change of intracellular lipid environment. We analyze the lipid compositions of the SV membrane by mass-spectrometry-based lipidomic profiling, and further reveal that VAMP2 forms distinctive conformations in different membrane regions. In contrast to the non-raft region, the membrane region of cholesterol-rich lipid raft markedly weakens the membrane association of VAMP2 SNARE motif, which releases the SNARE motif and facilitates the SNARE assembly. Our work reveals the regulation of different membrane regions on VAMP2 structure and sheds light on the spatial regulation of SNARE assembly.

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells.

Current healthcare practices are reactive and use limited physiological and clinical information, often collected months or years apart. Moreover, the discovery and profiling of blood biomarkers in clinical and research settings are constrained by geographical barriers, the cost and inconvenience of in-clinic venepuncture, low sampling frequency and the low depth of molecular measurements. Here we describe a strategy for the frequent capture and analysis of thousands of metabolites, lipids, cytokines and proteins in 10 μl of blood alongside physiological information from wearable sensors. We show the advantages of such frequent and dense multi-omics microsampling in two applications: the assessment of the reactions to a complex mixture of dietary interventions, to discover individualized inflammatory and metabolic responses; and deep individualized profiling, to reveal large-scale molecular fluctuations as well as thousands of molecular relationships associated with intra-day physiological variations (in heart rate, for example) and with the levels of clinical biomarkers (specifically, glucose and cortisol) and of physical activity. Combining wearables and multi-omics microsampling for frequent and scalable omics may facilitate dynamic health profiling and biomarker discovery.