The conformational landscape of human transthyretin revealed by cryo-EM.

Transthyretin (TTR) is a natively tetrameric thyroxine transporter found in blood and cerebrospinal fluid whose misfolding and aggregation causes transthyretin amyloidosis. A rational drug design campaign identified the small molecule tafamidis (Vyndaqel/Vyndamax) as an effective stabilizer of the native TTR fold, and this aggregation inhibitor is regulatory agency-approved for the treatment of TTR amyloidosis. Despite 50 years of structural studies on TTR and this triumph of structure-based drug design, there remains a notable dearth of structural information available to understand ligand binding allostery and amyloidogenic TTR unfolding intermediates. We used single-particle cryo-electron microscopy (cryo-EM) to investigate the conformational landscape of this 55 kiloDalton tetramer in the absence and presence of one or two ligands, revealing inherent asymmetries in the tetrameric architecture and previously unobserved conformational states. These findings provide critical mechanistic insights into negatively cooperative ligand binding and the structural pathways responsible for TTR amyloidogenesis. This study underscores the capacity of cryo-EM to provide new insights into protein structures that have been historically considered too small to visualize and to identify pharmacological targets suppressed by the confines of the crystal lattice, opening uncharted territory in structure-based drug design.

Aß Amyloid Scaffolds the Accumulation of Matrisome and Additional Proteins in Alzheimer's Disease.

We report a highly significant correlation in brain proteome changes between Alzheimers disease (AD) and CRND8 APP695NL/F transgenic mice. However, integrating protein changes observed in the CRND8 mice with co-expression networks derived from human AD, reveals both conserved and divergent module changes. For the most highly conserved module (M42, matrisome) we find many proteins accumulate in plaques, cerebrovascular amyloid (CAA), dystrophic processes, or a combination thereof. Overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), in CRND8 mice brains leads to increased accumulation of A ß ; in plaques and in CAA; further, recombinant MDK and PTN enhance A ß ; aggregation into amyloid. Multiple M42 proteins, annotated as heparan sulfate binding proteins, bind to fibrillar A ß 42 and a non-human amyloid fibril in vitro. Supporting this binding data, MDK and PTN co-accumulate with transthyretin (TTR) amyloid in the heart and islet amyloid polypeptide (IAPP) amyloid in the pancreas. Our findings establish several critical insights. Proteomic changes in modules observed in human AD brains define an A ß ; amyloid responsome that is well conserved from mouse model to human. Further, distinct amyloid structures may serve as scaffolds, facilitating the co-accumulation of proteins with signaling functions. We hypothesize that this co-accumulation may contribute to downstream pathological sequalae. Overall, this contextualized understanding of proteomic changes and their interplay with amyloid deposition provides valuable insights into the complexity of AD pathogenesis and potential biomarkers and therapeutic targets.

Transthyretin Aggregation Pathway toward the Formation of Distinct Cytotoxic Oligomers.

Characterization of small oligomers formed at an early stage of amyloid formation is critical to understanding molecular mechanism of pathogenic aggregation process. Here we identified and characterized cytotoxic oligomeric intermediates populated during transthyretin (TTR) aggregation process. Under the amyloid-forming conditions, TTR initially forms a dimer through interactions between outer strands. The dimers are then associated to form a hexamer with a spherical shape, which serves as a building block to self-assemble into cytotoxic oligomers. Notably, wild-type (WT) TTR tends to form linear oligomers, while a TTR variant (G53A) prefers forming annular oligomers with pore-like structures. Structural analyses of the amyloidogenic intermediates using circular dichroism (CD) and solid-state NMR reveal that the dimer and oligomers have a significant degree of native-like ß-sheet structures (35-38%), but with more disordered regions (~60%) than those of native TTR. The TTR variant oligomers are also less structured than WT oligomers. The partially folded nature of the oligomeric intermediates might be a common structural property of cytotoxic oligomers. The higher flexibility of the dimer and oligomers may also compensate for the entropic loss due to the oligomerization of the monomers.

The Diflunisal Trial: study accrual and drug tolerance.

Familial amyloidotic polyneuropathy (FAP) is a protein folding disorder that induces neuropathy and cardiomyopathy, leading to death within 7-15 years after onset of clinical disease. In vitro, small ligands binding the thyroid hormone docking site stabilize tetrameric transthyretin, inhibiting amyloid fibril formation. We undertook a randomized, placebo-controlled clinical trial to determine whether diflunisal, a well-known non-steroidal anti-inflammatory drug (NSAID) alters neurologic disease progression in FAP. We enrolled 130 subjects with wide age and FAP mutation representation. To date, few recognized complications of NSAIDs have occurred in the study cohort. Data collection will be completed by November 2012.

Crystal structure of human triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.47 A.

The triggering receptor expressed on myeloid cells (TREM) family of single extracellular immunoglobulin receptors includes both activating and inhibitory isoforms whose ligands are unknown. TREM-1 activation amplifies the Toll-like receptor initiated responses to invading pathogens allowing the secretion of pro-inflammatory chemokines and cytokines. Hence, TREM-1 amplifies the inflammation induced by both bacteria and fungi, and thus represents a potential therapeutic target. We report the crystal structure of the human TREM-1 extracellular domain at 1.47 A resolution. The overall fold places it within the V-type immunoglobulin domain family and reveals close homology with Ig domains from antibodies, T-cell receptors and other activating receptors, such as NKp44. With the additional use of analytical ultracentrifugation and 1H NMR spectroscopy of both human and mouse TREM-1, we have conclusively demonstrated the monomeric state of this extracellular ectodomain in solution and, presumably, of the TREM family in general.