The crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE), both with a transition state analog (MgADP x AIF4-) in the active site, have been determined to 2.9 A and 3.5 A resolution, respectively. The MDE structure with an ATP analog (MgADP x BeFx) was also determined to 3.6 A resolution. In all three structures, a domain of the C-terminal region, the "converter," is rotated approximately 70 degrees from that in nucleotide-free skeletal subfragment 1 (S1). We have found that the MDE-BeFx and MDE-AIF4- structures are almost identical, consistent with the fact that they both bind weakly to actin. A comparison of the lever arm positions in MDE-AIF4- and in nucleotide-free skeletal S1 shows that a potential displacement of approximately 10 nm can be achieved during the power stroke.

The induced pluripotent stem (iPS) cell field promises a new era for in vitro disease modeling. However, identifying innate cellular pathologies, particularly for age-related neurodegenerative diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved proteotoxic mechanisms from yeast to human to discover and reverse phenotypic responses to α-Synuclein (αSyn), a key protein involved in Parkinson’s disease (PD). We generated cortical neurons from iPS cells of patients harboring αSyn mutations, who are at high risk of developing PD dementia. Genetic modifiers from unbiased screens in a yeast model of αSyn toxicity led to identification of early pathogenic phenotypes in patient neurons. These included nitrosative stress, accumulation of ER-associated degradation (ERAD) substrates and ER stress. A small molecule identified in a yeast screen, and the ubiquitin ligase Nedd4 it activates, reversed pathologic phenotypes in these neurons.

Highlights d aS impacts lipid homeostasis, triggering excess oleic acid (OA) and diglycerides (DG) d Triglycerides and lipid droplets protect against toxicity by sequestering OA and DG d Stearoyl-CoA desaturase (SCD) inhibition rescues aS toxicity and neuron degeneration d SCD inhibition decreases aS inclusions and increases aS multimerization and solubility

Regulation of a variety of cellular contractile events requires that vertebrate smooth and non-muscle myosin II can achieve an ''off'' state. To examine the role of the myosin rod in this process, we determined the minimal size at which a myosin molecule is capable of regulation via light chain phosphorylation. Expressed smooth muscle myosin subfragments with as many as 100 amino acids of the coiledcoil rod sequence did not dimerize and were active independently of phosphorylation. To test whether dimerization per se restores regulation of ATPase activity, mutants were expressed with varying lengths of rod sequence, followed by C-terminal leucine zippers to stabilize the coiled-coil. Dimerization restored partial regulation, but the presence of a length of rod approximately equal to the myosin head was necessary to achieve a completely off state. Partially regulated short dimers could be converted into fully regulated molecules by addition of native rod sequence after the zipper. These results suggest that the myosin rod mediates specific interactions with the head that are required to obtain the completely inactive state of vertebrate smooth and non-muscle myosins. If these interactions are prohibited under cellular conditions, unphosphorylated crossbridges can slowly cycle.

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulinbinding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 ؎ 1.6 s ؊1 with a K app of ϳ1 M (37°C), and the velocity of actin movement was ϳ0.3 m/s (30°C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to ϳ70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s ؊1 ), and ATP hydrolysis occurs at ϳ200 s ؊1 . The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.Conventional muscle myosin II polymerizes into filaments and is designed to interact with actin as part of an ensemble, and the kinetic properties of myosins isolated from these tissues reflect this role. Their so-called "duty cycle," i.e. the length of time the myosin spends in a force-or motion-producing state, is relatively low compared with the overall cycle time determined by the ATPase activity. This feature allows for speed of contraction, and much of the cycle is spent in a state that is dissociated from actin. In contrast, unconventional myosins are nonfilamentous, and most of these classes of myosin will probably operate in much smaller groups or potentially even individually. Because of these different functional roles, the kinetic properties of these motors are expected to be quite different from the well characterized myosin IIs.Murine myosin V is a member of the class of unconventional myosins that is implicated in organelle movement and membrane trafficking based on a number of cellular and genetic studies. Mutations in murine myosin V result in a range of defects, from impaired pigment granule movement, resulting in a dilute coat color, to a lack of smooth endoplasmic reticulum in the dendritic spines of Purkinje cells, which may be the cause of the neurological defect that results in early postnatal death (reviewed in Ref. 1).Myosin V is particularly interesting from several points of view. It is a dimeric molecule that has an unusually long neck, three times that of myosin II. This region of the molecule has been proposed to act as a lever arm that ultimately results in relative sliding of actin and myosin. The neck region contains six IQ motifs that have the consensus sequence (IQXXIR-GXXXR) for binding of calmodulin or myosin light chains. Thus, the potential for calcium-dependent regulation of motor activity also exists fo...

Synucleinopathies, including Parkinson’s disease (PD), are associated with the misfolding and mistrafficking of alpha-synuclein (α-syn). Here, using an ascorbate peroxidase (APEX)-based labeling method combined with mass spectrometry, we defined a network of proteins in the immediate vicinity of α-syn in living neurons to shed light on α-syn function. This approach identified 225 proteins, including synaptic proteins, proteins involved in endocytic vesicle trafficking, the retromer complex, phosphatases and mRNA binding proteins. Many were in complexes with α-syn, and some were encoded by genes known to be risk factors for PD and other neurodegenerative diseases. Endocytic trafficking and mRNA translation proteins within this spatial α-syn map overlapped with genetic modifiers of α-syn toxicity, developed in an accompanying study (Khurana et al, 2016). Our data suggest that perturbation of these particular pathways is directly related to the spatial localization of α-syn within the cell. These approaches provide new avenues to systematically examine protein function and pathology in living cells.

Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson’s disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To “humanize” this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking/ER quality control and mRNA metabolism/translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control/function, metal ion transport, transcriptional regulation and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9, VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2 and EIF4G1/PARK18) were confirmed in patient iPS cell-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms, and may facilitate patient stratification for targeted therapy.

Regulatory light chain (RLC) phosphorylation is necessary to activate smooth muscle myosin, unlike constitutively active striated muscle myosins. Here we show that an actin-binding surface loop located at the 50/20-kDa junction contributes to this fundamental difference between myosins. Substitution of the native actin-binding loop of smooth muscle heavy meromyosin (HMM) with that from either skeletal or ␤-cardiac myosin caused the chimeric HMMs to become unregulated like the myosin from which the loop was derived. Dephosphorylated chimeric HMMs gained the ability to move actin in a motility assay and had 50 -70% of the actinactivated ATPase activity of phosphorylated wild-type HMM. Direct binding measurements showed that the affinity of HMM for actin in the presence of MgATP was unaffected by loop substitution; thus the rate of a step other than binding is increased. Phosphorylation of the chimeras did not lead to a higher V max than obtained for wild-type HMM. In the absence of actin, a foreign loop did not affect nucleotide trapping. Native regulated molecules have thus evolved a loop sequence which prevents rapid product release by actin when the RLC is dephosphorylated, thereby allowing activity to be controlled by RLC phosphorylation.Myosins differ from each other in how fast they can move actin, the rates of their actin-activated ATPases, and whether or not these activities are regulated. It has been recently suggested that a divergent surface loop at the actin-binding interface "tunes" the rate of phosphate release and thus sets the maximum velocity (V max ) for ATPase activity. A second loop, at the 25/50-kDa interface near the ATP-binding site, was proposed to control the rate of ADP release and thus be responsible for determining the velocity at which different myosins move actin (Spudich, 1994). The actin-activated ATPase activity of a chimeric Dictyostelium myosin containing the actin-binding loop from skeletal muscle myosin was 5-fold higher than wildtype Dictyostelium myosin, providing experimental evidence in support of the first part of this hypothesis (Uyeda et al., 1994). This study did not show if such a generalization would hold true for other myosin motors nor was the question of heavy chain sequences involved in regulation of activity addressed.The molecular step that is controlled by light chain phosphorylation in smooth muscle myosin is phosphate release from the active site (Sellers, 1985). It has been recently proposed that myosin is a "back door" enzyme, whereby the cleaved phosphate leaves via a cleft in the 50-kDa domain, instead of through the nucleotide-binding pocket from which it entered. Binding of actin is suggested to promote movement of the highly conserved P-loop near the active site so that phosphate can leave (Yount et al., 1995). The interaction of actin with myosin probably involves several steps; the first is thought to be a weak interaction between the N terminus of actin and the 50/20-kDa junction of myosin, followed by stronger interactions involving hydrophobic res...