The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A–retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (K d s ∼2 nM and ∼200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

In Saccharomyces cerevisiae, three chitin synthases have been detected. Chitin synthases I and II, the products of the CHS1 and CHS2 genes, respectively, are closely related proteins that require partial proteolysis for activity in vitro. In contrast, chitin synthase III is active in vitro without protease treatment, and three genes, CSD2 (= CAL1), CSD4 (= CAL2), and CAL3, are required for its activity. In the cell, the three enzymes have different functions. Chitin synthase I and II make only a small portion, < 10%, of the cellular chitin. In acidic media, chitin synthase I is required for normal budding. Chitin synthase II is required for normal morphology, septation, and cell separation. Chitin synthase III is required for the synthesis of 90% of the cellular chitin, including the chitin in the bud scars and lateral wall. Mutants defective in chitin synthase III are resistant to Calcofluor and Kluyveromyces lactis killer toxin, they lack alkali-insoluble glucan, and under certain circumstances, they are temperature-sensitive for growth. The available data suggest that many fungi have more than one chitin synthase and that these synthases are related to the S. cerevisiae CHS and CSD gene products.

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In Saccharomyces cerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. Using a novel screen, I have identified three mutations, designated csd2, csd3, and csd4, that reduce levels of chitin in vivo by as much as 10-fold without causing any obvious perturbation of cell division. The csd2 and csd4 mutants lack chitin synthase III activity in vitro, while csd3 mutants have wild-type levels of this enzyme. In certain genetic backgrounds, these mutations cause temperature-sensitive growth on rich medium; inclusion of salts or sorbitol bypasses this phenotype. Gene disruption experiments show that CSD2 is nonessential; a small amount of chitin, about 5% of the wild-type level, is detected in the disruptants. DNA sequencing indicates that the CSD2 protein has limited, but statistically significant, similarity to chitin synthase I and chitin synthase II. Other significant similarities are to two developmental proteins: the nodC protein from Rhizobium species and the DG42 protein ofXenopus laevis. The relationship between the nodC and CSD2 proteins suggests that nodC may encode an N-acetylglucosaminyltransferase that synthesizes the oligosaccharide backbone of the nodulation factor NodRm-1.In Saccharomyces cerevisiae, chitin plays an important role in cell division. A ring of chitin is made at the base of the emerging bud; following nuclear migration and cytokinesis, this ring is filled in to form a disc or primary septum that separates mother and daughter cells (10).Three chitin synthases have been detected in cell-free preparations from exponentially growing cells (6). Chitin synthase I and chitin synthase II are zymogens that require partial proteolysis for activity in vitro (10, 42); little or no activity can be detected in untreated membranes (9, 32). In contrast, chitin synthase III does not require proteolysis for activity (6,32). Because the synthesis of chitin is localized and occurs during a limited period of the cell cycle, it is likely that the activities of these enzymes are regulated temporally and spatially. The regulatory mechanisms used by the cell are not known.The structural genes for chitin synthase I and chitin synthase II, CHS1 (8) Botstein; it consists of Sau3A genomic DNA fragments inserted into the BamHI site of YEp24. Standard molecular cloning procedures (50) were used. The S. cerevisiae LEU2 gene, which was used as the selectable gene in the disruption experiments, was isolated from the plasmids YEp13 and pJH-L2. The latter is a derivative of pUC18 that has a 2.9-kb BglII fragment containing LEU2 inserted into the BamHI site; this plasmid was generously provided by J. Hill (Carnegie-Mellon University). A leuB mutant strain of Escherichia coli was used to identify recombinant plasmids containing LEU2.Media and growth conditions. The following media (45)