The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes. P arkinson disease (PD) is the most common movement disorder, currently affecting approximately 2% of the population older than age 60 y. Prominent neuropathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain (1) and the presence of α-syn-containing intracellular inclusions: Lewy bodies (LBs) and Lewy neurites (2). α-Syn, a 140-aa protein physiologically found in presynaptic terminals of neurons, is the major fibrillar protein in LBs and Lewy neurites in sporadic and inherited PD. Moreover, point mutations (A53T, A30P, E46K) and gene multiplications of human WT (hWT) α-syn are related to rare familial autosomal-dominant forms of early-onset PD (3-6), suggesting that increased gene dosage and aberrant protein structure may accelerate disease onset and progression.Recent reports indicate that the accumulation of α-syn can result in the formation of intermediate-state oligomers, and oligomers of different shapes and sizes have been described (7-10). These oligomers interact with lipids, disrupt membranes (7,8), and cause cell death in vitro (10, 11) and in nonmammalian models, such as Caenorhabditis elegans and Drosophila melanogaster (12). However, we are aware of no previous direct in vivo demonstration of the toxicity of α-syn oligomers in mammals.We aim to establish a model that allows specific testing of the effects of α-syn oligomerization in vitro and in vivo. To elucidate the causal structure-toxicity relationship of these oligomeric protein assemblies in a mammalian system, we designed "conformation-trapped" mutants based on structural modeling of α-syn fibrils (13, 14). Structurally, amyloid fibrils of α-syn are composed of cross-β-sheets (15). Residues from approximately 30 to 110 of α-syn form the core of the fibrils, whereas the approximately 30 N-terminal residues are heterogeneous and the approximately 30 C-terminal residues are flexible (13,14,16,17). Based on our structural model, recently developed from NMR data, the core of α-syn fibrils comprises five β-strands reminiscent of a five-layered "β-sandwich" (14). Several loops adjacent to and between the strands ar...

Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAF-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRAS). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

SUMMARY X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase apoptotic activity. XIAP also functions as an E3 ubiquitin ligase, targeting caspases for degradation. However, molecular pathways controlling XIAP activities remain unclear. Here we report that nitric oxide (NO) reacts with XIAP by S-nitrosylating its RING domain (forming SNO-XIAP), thereby inhibiting E3 ligase and antiapoptotic activity. NO-mediated neurotoxicity and caspase activation have been linked to several neurodegenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. We find significant SNO-XIAP formation in brains of patients with these diseases, implicating this reaction in the etiology of neuronal damage. Conversely, S-nitrosylation of caspases is known to inhibit apoptotic activity. Unexpectedly, we find that SNO-caspase transnitrosylates (transfers its NO group) to XIAP, forming SNO-XIAP, and thus promotes cell injury and death. These findings provide unique insights into the regulation of caspase activation in neurodegenerative disorders mediated, at least in part, by nitrosative stress.

K + channels conduct and regulate K + flux across the cell membrane. Several crystal structures and biophysical studies of tetrameric ion channels have revealed many of the structural details of ion selectivity and gating. A narrow pore lined with four arrays of carbonyl groups is responsible for ion selectivity, whereas a conformational change of the four inner transmembrane helices (TM2) is involved in gating. We used NMR to examine full-length KcsA, a prototypical K + channel, in its open, closed and intermediate states. These studies reveal that at least two conformational states exist both in the selectivity filter and near the C-terminal ends of the TM2 helices. In the ion-conducting open state, we observed rapid structural exchange between two conformations of the filter, presumably of low and high K + affinity, respectively. Such measurements of millisecond-timescale dynamics reveal the basis for simultaneous ion selection and gating.The simplest structural definition of ion channel gating is the ability to alternate, in a signaldependent way, between states that selectively permit the flow of ions (open) or do not (closed). In the closed state, generally a default state, K + channels inhibit ion flow with high efficacy. In the open state, two opposing tasks must be carried out near the diffusion limit: ion selection and permeation 1 . Energetically, ion selection requires spatial coordination that favors K + over other cations, whereas permeation requires the rapid release of the cation from these recognition sites. This 'hold-and-release' feature underlies the unique structural and dynamical properties of the channel.Crystal structures of K + channels of prokaryotic origin (KcsA 2 , MthK 3 , KirBac1.1 (ref. 4), KvAP 5 and NaK 6 ) have revealed a universal structural framework for these tasks. To explain ion selectivity, the structures show a narrow filter composed of four successive layers of main chain carbonyl oxygen atoms, which act as surrogate waters for the dehydrated K + ions (1.35-Å radius) as they pass through the filter. This unique spatial geometry results from the signature filter amino acid sequence, Gly-Tyr-Gly, in which the two glycines enable positive φ/ψ angles of the protein backbone so that the K + -coordinating carbonyl oxygens can all point inward to the pore axis simultaneously 7 to perform ion selection 8,9 . For gating of the channel, the pore-lining C-terminal ends of the inner AUTHOR CONTRIBUTIONSK.A.B. and C.T. prepared KcsA; K.A.B., C.T., W.K. and R.R. collected and analyzed NMR data; K.A.B., C.T., S.C. and R.R. contributed to scientific discussions and prepared the manuscript. Fig. 3 online), we have obtained B85% of the sequential backbone assignments of full-length KcsA(tox) at pH 7 (Fig. 1). A residue-specific secondary structure analysis based on positive 13 Cα chemical shift deviations from a random coil (Fig. 1a), amide-H 2 O exchange ( Supplementary Fig. 4b online) and protein-detergent NOEs ( Supplementary Fig. 4a) revealed the presence of the following he...