Many chronic human diseases, including multiple neurodegenerative diseases, are associated with deleterious protein aggregates, also called protein amyloids. One common therapeutic strategy is to develop protein aggregation inhibitors that can slow down, prevent, or remodel toxic amyloids. Natural products are a major class of amyloid inhibitors, and several dozens of natural product-based amyloid inhibitors have been identified and characterized in recent years. These plant- or microorganism-extracted compounds have shown significant therapeutic potential from in vitro studies as well as in vivo animal tests. Despite the technical challenges of intrinsic disordered or partially unfolded amyloid proteins that are less amenable to characterizations by structural biology, a significant amount of research has been performed, yielding biochemical and pharmacological insights into how inhibitors function. This review aims to summarize recent progress in natural product-based amyloid inhibitors and to analyze their mechanisms of inhibition in vitro. Major classes of natural product inhibitors and how they were identified are described. Our analyses comprehensively address the molecular interactions between the inhibitors and relevant amyloidogenic proteins. These interactions are delineated at molecular and atomic levels, which include covalent, non-covalent, and metal-mediated mechanisms. In vivo animal studies and clinical trials have been summarized as an extension. To enhance natural product bioavailability in vivo, emerging work using nanocarriers for delivery has also been described. Finally, issues and challenges as well as future development of such inhibitors are envisioned.

γδ T cells play important roles in bridging innate and adaptive immunity, but their recognition mechanisms remain poorly understood. Human γδ T cells of the V δ 1 subset predominate in intestinal epithelia and respond to MICA and MICB (MHC class I chain-related, A and B; MIC) self-antigens, mediating responses to tumorigenesis or viral infection. The crystal structure of an MIC-reactive V δ 1 γδ T-cell receptor (TCR) showed expected overall structural homology to antibodies, αβ, and other γδ TCRs, but complementary determining region conformations and conservation of V δ 1 use revealed an uncharacteristically flat potential binding surface. MIC, likewise, serves as a ligand for the activating immunoreceptor natural killer group 2, D (NKG2D), also expressed on γδ T cells. Although MIC recognition drives both the TCR-dependent stimulatory and NKG2D-dependent costimulatory signals necessary for activation, interaction analyses showed that MIC binding by the two receptors was mutually exclusive. Analysis of relative binding kinetics suggested sequential recognition, defining constraints for the temporal organization of γδ T-cell/target cell interfaces.biacore | crystallography D espite comprising only a small fraction (2-3%) of the total human T-cell population, γδ T cells contribute to immune surveillance by elimination of malignant cells and recognition of mucosal and peripheral blood-borne pathogens (1). However, in contrast to αβ T-cell receptors (TCRs), which require antigen processing and subsequent presentation by MHC molecules, γδ TCRs are believed to recognize antigens directly (2-4). Little is known about the details of γδ TCR ligand recognition mechanisms, because receptor-ligand pairs for this class of immunoreceptors have been difficult to identify. Unlike αβ TCRs, for which there are dozens of 3D structures available that provide a wealth of detailed information (5), there are only three γδ TCR structures currently known: an isolated human V δ 3 domain of unknown ligand specificity [ES204; Protein Data Bank (PDB) code 1TVD], the intact but ligand-free ectodomain of a human V γ 9V δ 2 TCR G115 (the predominant combination in the peripheral blood; PDB code 1HXM), and the murine γδ TCR G8 bound to its nonclassical MHC class I ligand, H-2T22 (PDB 1YPZ) (6-8). These structures are highly informative, showing, for instance, the expected high level of structural homology between αβ and γδ TCRs. However, they provide little detail about how human V δ 1 TCRs recognize ligands or about γδ TCR recognition in general. The murine G8:T22 complex structure, although showing an unusual recognition strategy dominated almost to exclusivity by a single complementary determining region (CDR) CDR3δ, may or may not recapitulate aspects of general γδ TCR ligand recognition. Additional structures of human γδ TCRs, with or without ligand, are, therefore, needed to elucidate molecular recognition mechanisms defining γδ TCR specificity, which are needed to fully understand γδ T cell-mediated immune responses.Human γδ T cells are...

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How insulin binds to and activates the insulin receptor has long been the subject of speculation. Of particular interest are invariant phenylalanine residues at consecutive positions in the B chain (residues B24 and B25). Sites of mutation causing diabetes mellitus, these residues occupy opposite structural environments: Phe(B25) projects from the surface of insulin, whereas Phe(B24) packs against the core. Despite these differences, site-specific cross-linking suggests that each contacts the insulin receptor. Photoactivatable derivatives of insulin containing respective p-azidophenylalanine substitutions at positions B24 and B25 were synthesized in an engineered monomer (DKP-insulin). On ultraviolet irradiation each derivative cross-links efficiently to the receptor. Packing of Phe(B24) at the receptor interface (rather than against the core of the hormone) may require a conformational change in the B chain. Sites of cross-linking in the receptor were mapped to domains by Western blot. Remarkably, whereas B25 cross-links to the C-terminal domain of the alpha subunit in accord with previous studies (Kurose, T., et al. (1994) J. Biol. Chem. 269, 29190-29197), the probe at B24 cross-links to its N-terminal domain (the L1 beta-helix). Our results demonstrate that consecutive residues in insulin contact widely separated sequences in the receptor and in turn suggest a revised interpretation of electron-microscopic images of the complex. By tethering the N- and C-terminal domains of the extracellular alpha subunit, insulin is proposed to stabilize an active conformation of the disulfide-linked transmembrane tyrosine kinase.

Amyloid formation of the 37-residue amylin is involved in the pathogenesis of type 2 diabetes and, potentially, diabetes-induced neurological deficits. Numerous flavonoids exhibit inhibitory effects against amylin amyloidosis, but the mechanisms of inhibition remain unclear. Screening a library of natural compounds uncovered a potent lead compound, the flavone baicalein. Baicalein inhibits amylin amyloid formation and reduces amylin-induced cytotoxicity. Analogue analyses demonstrated, for the first time, key roles of the vicinal hydroxyl groups on the A-ring. We provided mass spectrometric evidence that incubating baicalein and amylin leads to their conjugation, consistent with a Schiff base mechanism.

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Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic ␤-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain ␤-strand (residues B24 -B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the ␣-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of a ␤-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanningmutagenesiscandecodetheactiveconformationofaprotein and so illuminate cryptic constraints underlying its evolution.

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