No abstract

MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.

Mutation-induced aggregation of the dimeric enzyme Cu, Zn superoxide dismutase 1 (SOD1) has been implicated in the familial form of the disease amyotrophic lateral sclerosis, but the mechanism of aggregation is not known. Here, we show that in vitro SOD1 aggregation is a multistep reaction that minimally consists of dimer dissociation, metal loss from the monomers, and oligomerization of the apo-monomers:where Dholo, Mholo, Mapo, and A are the holo-dimer, holo-monomer, apo-monomer, and aggregate, respectively. Under aggregationpromoting conditions (pH 3.5), the rate and equilibrium constants corresponding to each step are: A myotrophic lateral sclerosis (ALS) involves selective motor neuron death in the brain and spinal cord (1-6), initiating a progressive paralysis in midlife. Point mutations in the cytoplasmic homodimeric enzyme Cu, Zn superoxide dismutase 1 [SOD1, molecular mass (M m ) Ϸ 32 kDa] were identified as the primary cause of Ϸ20% cases of the familial form of ALS (FALS) (7,8) in contrast with the sporadic form. Approximately 90 distinct FALS mutations are known (9). The toxic gain-of-function of the mutants is believed to be associated with either intracellular misfolding and aggregation or oxidative damage caused by mutant SOD1-catalyzed aberrant reactions, although the two scenarios may not be mutually exclusive (10, 11). The aggregation hypothesis is supported by the observations that in both mice and cell culture models death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD1 (12-15), SOD1 knockout mice do not develop motor neuron disease (16), and even Cu-depleted SOD1 mutants cause the disease in mice (17). Although aggregation of SOD1 has also been found in some cases of sporadic ALS (18)(19)(20), the sporadic disease is believed to have a different molecular basis (1). FALS and SOD1-linked sporadic ALS may belong to the general class of protein conformational disorders in which perturbation of protein folding leads to a relatively higher population of misfolded or partially folded (21) protein molecules, which then aggregate into regular (22) structured fibrils. Toxicity may arise because of the saturation of the cellular chaperone machinery by misfolded or aggregated SOD1 molecules (23).ALS is an age-related disease occurring in midlife or later, and it is not surprising that the in vitro aggregation of SOD1 solutions under physiological conditions is slow. Therefore, it is common to study SOD1 aggregation by perturbing the environmental conditions, lowering the pH to 3.5 (24), adding mild denaturants such as trifluoroethanol, or using heat treatment (25), such that disease-like aggregates can be detected on experimental time scales. Aggregation-prone FALS mutations and͞or loss of metals also decrease SOD1 stability (26,27), indicating that exposure to nonphysiological conditions, such as low pH, mimics the effect of mutation by similarly lowering the barrier for SOD1 aggregation. Although the rate of aggregation can be enhanced in vitro, the molecula...

Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the K d . This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nM rather than 212 nM at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. Familial amyotrophic lateral sclerosis (FALS)4 is the hereditary form of amyotrophic lateral sclerosis, a fatal disease characterized by progressive motor neuron loss (1). A subset of FALS is caused by mutations in the gene encoding homodimeric Cu/Zn-superoxide dismutase (SOD1), which forms intraneuronal aggregates (2). Although SOD1 aggregation is involved in SOD1-mediated FALS, it is generally believed that the functional properties of the enzyme are not related to the toxic gain of function imparted by mutations in SOD1 (3). However, the discovery of roles for SOD1 in the regulation of the cellular phosphorylation balance (4) and redox state (5) provides additional avenues for connecting the cellular role of SOD1 to FALS. The classical studies of SOD1 were generally performed using bovine erythrocyte SOD1 or recombinant human SOD1. Although recombinant methods are widely used to produce SOD1 mutants, a disadvantage of studying recombinant SOD1 is the absence of potentially important post-translational modifications present in human tissues. The initial SOD1 crystal structure was solved using bovine erythrocyte SOD1 (6) and no structure of human erythrocyte SOD1 is available. Here we report results using human erythrocyte SOD1 rather than the recombinant enzyme and find that the native enzyme features a consistent pattern of post-translational modifications. Using a combination of "bottom-up" and "top-down" mass spectrometry (MS) approaches, we show that SOD1 isolated from human erythrocytes is post-translationally phosphorylated and glutathionylated. These modifications occur near the SOD1 dimer interface. Because monomer formation is thought to be the first intermediate leading to SOD1 aggregation (7, 8), we tested the dimer stability of modified SOD1 found, as expected, that glutathionylation promotes the formation of SOD1 monomer. EXPERIMEN...

Abstract. The standard free energy for hydrolysis of the GTP analogue guanylyl-(a,b)-methylenediphosphonate (GMPCPP), which is -5.18 kcal in solution, was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtubules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that the majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described here were obtained from video microscopy measurements of the kinetics of assembly and disassembly of GMPCPP-microtubules and GMPCPmicrotubules. It was possible to study GMPCPPmicrotubules since GMPCPP is not hydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassium) Pipes buffer with glycerol, which reduced the halftime for GMPCPP hydrolysis from >10 h to ",,10 min. The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s -1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.T HE hallmark for energy-transducing systems such as myosin (Bagshaw and Trentham, 1973), ion-transporting ATPases (Taniguchi, and Post, 1975;Pickart and Jencks, 1984), the mitochondrial ATPase (Grubmeyer et al., 1982) and chloroplast coupling factor (Feldman and Sigman, 1982) is a near-zero free energy for hydrolysis of bound nucleotide triphosphate (NTP)L Negligible free energy is released during NTP hydrolysis since this is stored in the protein conformation until useful work can be done. We have used the hydrolyzable GTP analogue GMPCPP, which contains a methylene linkage between the alpha and beta phosphates, to determine the free energy for hydrolysis of microtubule-bound NTP. We found that the majority of this free energy is stored in the microtubule lattice, so that the standard free energy for hydrolysis is near zero. This stored energy can do work, as in the NTP-independent movement of chromosomes in a reaction coupled to microtubule disassembly (Koshland et al., 1988;Coue et al., 1991 bules parallels the behavior of several energy-transducing systems.In addition to these equilibrium studies, we have characterized the dynamics of microtubules formed with GMPCPP. The recent observation that this substance is not hydrolyzed in microtubules (Hyman et al., 1992) suggested its use in determining the role of the gamma phosphate moiety in microtubule dynamics. Our rationale was that if conditions could be found for forming microtubules containing GMPCP, it would be possible to determine the kinetic behavior of microtubules with a cognate pair of bound nucleotide tri-and diphosphates. Such a comparison was not possible with GTP analo...

Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly (catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average of 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25-to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25-to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCPP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 or 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of the rate constant for dissociation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microtubules, now found to be 0.118 s-1, has allowed a better estimate of the standard free energy for hydrolysis of GMPCPP in a microtubule and release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as previously reported.

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1WT dimers, increasing the equilibrium dissociation constant Kd to ~10−20 μM, comparable to that of the aggressive A4V mutant. SOD1A4V is further destabilized by glutathionylation, experiencing an ~30-fold increase in Kd. Dissociation kinetics of glutathionylated SOD1WT and SOD1A4V are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1I112T has a modestly increased dissociation rate but no change in Kd when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.