The receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling molecule in the programmed necrosis (necroptosis) pathway. This pathway plays important roles in a variety of physiological and pathological conditions, including development, tissue damage response, and antiviral immunity. Here, we report the identification of a small molecule called (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide--hereafter referred to as necrosulfonamide--that specifically blocks necrosis downstream of RIP3 activation. An affinity probe derived from necrosulfonamide and coimmunoprecipitation using anti-RIP3 antibodies both identified the mixed lineage kinase domain-like protein (MLKL) as the interacting target. MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis. Treating cells with necrosulfonamide or knocking down MLKL expression arrested necrosis at a specific step at which RIP3 formed discrete punctae in cells. These findings implicate MLKL as a key mediator of necrosis signaling downstream of the kinase RIP3.

Programmed necrotic cell death induced by the tumor necrosis factor alpha (TNF-α) family of cytokines is dependent on a kinase cascade consisting of receptor-interacting kinases RIP1 and RIP3. How these kinase activities cause cells to die by necrosis is not known. The mixed lineage kinase domain-like protein MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. RIP3 phosphorylates MLKL at the T357 and S358 sites. Reported here is the development of a monoclonal antibody that specifically recognizes phosphorylated MLKL in cells dying of this pathway and in human liver biopsy samples from patients suffering from drug-induced liver injury. The phosphorylated MLKL forms an oligomer that binds to phosphatidylinositol lipids and cardiolipin. This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death.

SUMMARY MLKL is crucial for necroptosis, permeabilizing membranes through its N-terminal region upon phosphorylation of its kinase-like domain by RIP3. However, the mechanism underlying membrane permeabilization is unknown. The solution structure of the MLKL N-terminal region determined by NMR spectroscopy reveals a four-helix bundle with an additional helix at the top that is likely key for MLKL function, and a sixth, C-terminal helix that interacts with the top helix and with a poorly packed interface within the four-helix bundle. Fluorescence spectroscopy measurements indicate that much of the four-helix bundle inserts into membranes, but not the C-terminal helix. Moreover, we find that the four-helix bundle is sufficient to induce liposome leakage and that the C-terminal helix inhibits this activity. These results suggest that the four-helix bundle mediates membrane breakdown during necroptosis and that the sixth helix acts as a plug that prevents opening of the bundle and is released upon RIP3 phosphorylation.

KChIPs coassemble with pore-forming Kv4 alpha subunits to form a native complex in the brain and heart and regulate the expression and gating properties of Kv4 K(+) channels, but the mechanisms underlying these processes are unknown. Here we report a co-crystal structure of the complex of human Kv4.3 N-terminus and KChIP1 at a 3.2-A resolution. The structure reveals a unique clamping action of the complex, in which a single KChIP1 molecule, as a monomer, laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner, forming an octamer. The proximal N-terminal peptide of Kv4.3 is sequestered by its binding to an elongated groove on the surface of KChIP1, which is indispensable for the modulation of Kv4.3 by KChIP1, and the same KChIP1 molecule binds to an adjacent T1 domain to stabilize the tetrameric Kv4.3 channels. Taken together with biochemical and functional data, our findings provide a structural basis for the modulation of Kv4 by KChIPs.

Preventing aggregation of amyloid beta (Aβ) peptides is a promising strategy for the treatment of Alzheimer’s disease (AD), and gold nanoparticles have previously been explored as a potential anti-Aβ therapeutics. Here we design and prepare 3.3 nm L- and D-glutathione stabilized gold nanoparticles (denoted as L3.3 and D3.3, respectively). Both chiral nanoparticles are able to inhibit aggregation of Aβ42 and cross the blood-brain barrier (BBB) following intravenous administration without noticeable toxicity. D3.3 possesses a larger binding affinity to Aβ42 and higher brain biodistribution compared with its enantiomer L3.3, giving rise to stronger inhibition of Aβ42 fibrillation and better rescue of behavioral impairments in AD model mice. This conjugation of a small nanoparticle with chiral recognition moiety provides a potential therapeutic approach for AD.

The WD40 repeat protein WDR5 specifically associates with the K4-methylated histone H3 in human cells. To investigate the structural basis for this specific recognition, we have determined the structure of WDR5 in complex with a dimethylated H3-K4 peptide at 1.9 A resolution. Unlike the chromodomain that recognizes the methylated H3-K4 through a hydrophobic cage, the specificity of WDR5 for methylated H3-K4 is conferred by the nonconventional hydrogen bonds between the two zeta-methyl groups of the dimethylated Lys4 and the carboxylate oxygen of Glu322 in WDR5. The three amino acids Ala-Arg-Thr preceding Lys4 form most of the specific contacts with WDR5, with Ala1 forming intermolecular hydrogen bonds and salt bridges, and the side chain of Arg2 inserting into the central channel of WDR5. Both structural and biochemical studies presented here suggest another mode of recognition for the methylated histone tail.

Receptor-interacting protein kinase 3, RIP3, and a pseudokinase mixed lineage kinase-domain like protein, MLKL, constitute the core components of the necroptosis pathway, which causes programmed necrotic death in mammalian cells. Latent RIP3 in the cytosol is activated by several upstream signals including the related kinase RIP1, which transduces signals from the tumor necrosis factor (TNF) family of cytokines. We report here that RIP3 activation following the induction of necroptosis requires the activity of an HSP90 and CDC37 cochaperone complex. This complex physically associates with RIP3. Chemical inhibitors of HSP90 efficiently block necroptosis by preventing RIP3 activation. Cells with knocked down CDC37 were unable to respond to necroptosis stimuli. Moreover, an HSP90 inhibitor that is currently under clinical development as a cancer therapy was able to prevent systemic inflammatory response syndrome in rats treated with TNF-α. HSP90 and CDC37 cochaperone complex-mediated protein folding is thus an important part of the RIP3 activation process during necroptosis.

Atmospheric CO2 is an important environmental cue that regulates several types of animal behavior. In mice, CO 2 responses of the olfactory sensory neurons (OSNs) require the activity of carbonic anhydrase to catalyze the conversion of CO 2 to bicarbonate and the opening of cGMP-sensitive ion channels. However, it remains unknown how the enhancement of bicarbonate levels results in cGMP production. Here, we show that bicarbonate activates cGMP-producing ability of guanylyl cyclase-D (GC-D), a membrane GC exclusively expressed in the CO 2-responsive OSNs, by directly acting on the intracellular cyclase domain of GC-D. Also, the molecular mechanism for GC-D activation is distinct from the commonly believed model of ''release from repression'' for other membrane GCs. Our results contribute to our understanding of the molecular mechanisms of CO 2 sensing and suggest diverse mechanisms of molecular activation among membrane GCs.carbonic anhydrase ͉ CNG channels ͉ necklace glomeruli ͉ transduction