α-Synuclein (aSyn) fibrillar polymorphs have distinct in vitro and in vivo seeding activities, contributing differently to synucleinopathies. Despite numerous prior attempts, how polymorphic aSyn fibrils differ in atomic structure remains elusive. Here, we present fibril polymorphs from the full-length recombinant human aSyn and their seeding capacity and cytotoxicity in vitro. By cryo-electron microscopy helical reconstruction, we determine the structures of the two predominant species, a rod and a twister, both at 3.7 Å resolution. Our atomic models reveal that both polymorphs share a kernel structure of a bent β-arch, but differ in their inter-protofilament interfaces. Thus, different packing of the same kernel structure gives rise to distinct fibril polymorphs. Analyses of disease-related familial mutations suggest their potential contribution to the pathogenesis of synucleinopathies by altering population distribution of the fibril polymorphs. Drug design targeting amyloid fibrils in neurodegenerative diseases should consider the formation and distribution of concurrent fibril polymorphs.

Subcellular membrane-less organelles consist of proteins with low complexity domains. Many of them, such as hnRNPA1, can assemble into both a polydisperse liquid phase and an ordered solid phase of amyloid fibril. The former mirrors biological granule assembly, while the latter is usually associated with neurodegenerative disease. Here, we observe a reversible amyloid formation of hnRNPA1 that synchronizes with liquid–liquid phase separation, regulates the fluidity and mobility of the liquid-like droplets, and facilitates the recruitment of hnRNPA1 into stress granules. We identify the reversible amyloid-forming cores of hnRNPA1 (named hnRACs). The atomic structures of hnRACs reveal a distinct feature of stacking Asp residues, which contributes to fibril reversibility and explains the irreversible pathological fibril formation caused by the Asp mutations identified in familial ALS. Our work characterizes the structural diversity and heterogeneity of reversible amyloid fibrils and illuminates the biological function of reversible amyloid formation in protein phase separation.

Inhibiting the interaction between ß-amyloid (Aß) and a neuronal cell surface receptor, LilrB2, has been suggested as a potential route for treating Alzheimer’s disease (AD). Supporting this approach, AD-like symptoms are reduced in mouse models following genetic depletion of the LilrB2 homolog. In its pathogenic, oligomeric state, Aß binds to LilrB2, triggering a pathway to synaptic loss. Here we identified the LilrB2 binding moieties of Aß ( 16 KLVFFA 21 ) and identified its binding site on LilrB2 from a crystal structure of LilrB2 immunoglobulin domains D1D2 complexed to small molecules that mimic phenylalanine residues. In this structure, we observed two pockets that can accommodate the phenylalanine sidechains of KLVFFA. These pockets were confirmed to be 16 KLVFFA 21 binding sites by mutagenesis. Rosetta docking revealed a plausible geometry for the Aß-LilrB2 complex and assisted with the structure-guided selection of small molecule inhibitors. These molecules inhibit Aß-LilrB2 interactions in vitro and on the cell surface and reduce Aß cytotoxicity, which suggests these inhibitors are potential therapeutic leads against AD.

BackgroundRepeated failure of drug candidates targeting Alzheimer’s disease (AD) in clinical trials likely stems from a lack of understanding of the molecular mechanisms underlying AD pathogenesis. Recent research has highlighted synergistic interactions between aggregated amyloid-β (Aβ) and tau proteins in AD, but the molecular details of how these interactions drive AD pathology remain elusive and speculative.MethodsHere, we test the hypothesis that Aβ potentiates intracellular tau aggregation, and show that oligomeric Aβ specifically exacerbates proteopathic seeding by tau. Using tau-biosensor cells, we show that treatment with sub-toxic concentrations of Aβ oligomers, but not monomers or fibrils, “primes” cells, making them more susceptible to tau seeding. The treatment with Aβ oligomers enhances intracellular tau aggregation in a dose-dependent manner when the cells are seeded with either recombinant or brain-derived tau fibrils, whereas little or no aggregation is observed in the absence of Aβ-oligomer priming.ResultsPriming by Aβ oligomers appears to be specific to tau, as α-synuclein seeding is unaffected by this treatment. Aβ oligomer-enhanced tau seeding also occurs in primary mouse neurons and human neuroblastoma cells. Using fluorescently labeled tau seeds, we find that treatment with Aβ oligomers significantly enhances the cellular uptake of tau seeds, whereas a known tau-uptake inhibitor blocks the effect of Aβ on tau uptake.ConclusionThe ability of Aβ to promote tau seeding suggests a specific and plausible mechanism by which extracellular Aβ initiates a deleterious cascade that is unique to AD. These data suggest that the Aβ-mediated potentiation of tau uptake into cells should also be taken into account when designing Aβ-targeted therapeutics.

Thymidylate kinase (TMPK) is a key enzyme for pyrimidine synthesis that catalyzes the phosphorylation of thymidine 5'-monophosphate (dTMP) in the presence of ATP and Mg(2+) to form thymidine 5'-diphosphate (dTDP), which is then converted to thymidine 5'-triphosphate (dTTP) by nucleoside-diphosphate kinase (NDK). TMPK has an important function in cell proliferation and its enzyme kinetics and related structures have been determined in various organisms. TMPK is well recognized as a potential drug target, with the most notable function being in the activation of anti-HIV nucleoside prodrugs. Recent studies have shown that TMPK is a validated target for antibiotic development against gram-positive bacterium of Staphylococcus aureus. In addition, inhibition of human TMPK increases the potential of anticancer agent doxorubicin toward colon cancer cells regardless of p53 status. Following the rapid expanding knowledge on TMPKs and the rising interests in TMPKs as a drug target, in this review we try to describe current research on TMPKs in various organisms of eukaryotes, prokaryotes and viruses and to provide information for designing new potential inhibitors against TMPKs.