Non-native soluble oligomers of Cu/Zn superoxide dismutase (SOD1) contain a conformational epitope linked to cytotoxicity in amyotrophic lateral sclerosis (ALS).

Soluble misfolded Cu/Zn superoxide dismutase (SOD1) is implicated in motor neuron death in amyotrophic lateral sclerosis (ALS); however, the relative toxicities of the various non-native species formed by SOD1 as it misfolds and aggregates are unknown. Here, we demonstrate that early stages of SOD1 aggregation involve the formation of soluble oligomers that contain an epitope specific to disease-relevant misfolded SOD1; this epitope, recognized by the C4F6 antibody, has been proposed as a marker of toxic species. Formation of potentially toxic oligomers is likely to be exacerbated by an oxidizing cellular environment, as evidenced by increased oligomerization propensity and C4F6 reactivity when oxidative modification by glutathione is present at Cys-111. These findings suggest that soluble non-native SOD1 oligomers, rather than native-like dimers or monomers, share structural similarity to pathogenic misfolded species found in ALS patients and therefore represent potential cytotoxic agents and therapeutic targets in ALS.

Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers.

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 SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 µM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) 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.

Ubiquitin editing enzyme UCH L1 and microtubule dynamics: implication in mitosis.

Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, western blot analysis of tubulin fractions after polymerization revealed the presence of a specific approximately 50 kDa band of UCH L1 (not the normal approximately 25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. The inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the approximately 50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins.

Conservation of structure and protein-protein interactions mediated by the secreted mycobacterial proteins EsxA, EsxB, and EspA.

Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (approximately 100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately alpha-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.

Modifications of superoxide dismutase (SOD1) in human erythrocytes: a possible role in amyotrophic lateral sclerosis.

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.

FALS mutations in Cu, Zn superoxide dismutase destabilize the dimer and increase dimer dissociation propensity: a large-scale thermodynamic analysis.

Mutations in the dimeric enzyme Cu, Zn superoxide dismutase (SOD1) leading to its aggregation are implicated in the toxicity in familial amyotrophic lateral sclerosis (FALS). We and others have previously shown that aggregation occurs by a pathway involving dimer dissociation, metal-loss from monomers and multimeric assembly of apo-SOD1 monomers. We postulate that FALS mutations cause enhanced aggregation by affecting one or more steps in the pathway, and computationally test this postulate for 75 known mis-sense FALS mutants of SOD1. Based on an extensive thermodynamic analysis of the stability of apo-dimer and apo-monomer forms of these mutants, we classify the mutations into the following groups: 70 out of 75 mutations in SOD1 lead to (i) decreased dimer stability, and/or (ii) increased dimer dissociation, compared to wild type, and four mutations lead to (iii) decreased monomer stability compared to wild type. Our results suggest that enhanced aggregation of SOD1 in FALS occurs due to an increased population of mutant SOD1 apo-monomers compared to wild type. The dissociation of multimeric proteins induced by diverse mutations may be a common theme in several human diseases.

The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis.

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: [reaction: see text], where D(holo), M(holo), M(apo), and A are the holo-dimer, holo-monomer, apo-monomer, and aggregate, respectively. Under aggregation-promoting conditions (pH 3.5), the rate and equilibrium constants corresponding to each step are: (i) dimer dissociation, Kd approximately 1 microM; k(off) approximately 1 x 10(-3) s(-1), k(on) approximately 1 x 10(3) M(-1).s(-1); (ii) metal loss, Km approximately 0.1 microM, km- approximately 1 x 10(-3)s(-1), km+ approximately 1 x 10(4) M(-1).s(-1); and (iii) assembly (rate-limiting step), k(agg) approximately 1 x 10(3) M(-1).s(-1). In contrast, under near-physiological conditions (pH 7.8), where aggregation is drastically reduced, dimer dissociation is less thermodynamically favorable: Kd approximately 0.1 nM, and extremely slow: k(off) approximately 3 x 10(-5) s(-1), k(on) approximately 3 x 10(5) M(-1).s(-1). Our results suggest that familial amyotrophic lateral sclerosis-linked SOD1 aggregation occurs by a mutation-induced increase in dimer dissociation and/or increase in apomonomer formation.

The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends.

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.

Concerning the chemical nature of tubulin subunits that cap and stabilize microtubules.

There is no definitive evidence on the nature of the cap at microtubule ends that is responsible for dynamic instability behavior. It was, therefore, of interest that steady-state microtubules assembled in 20 mM P(i) buffer and pulsed for 15-60 min with [gamma-(32)P]GTP contained approximately 26 [(32)P]P(i)/microtubule [Panda et al. (2002) Biochemistry 41, 1609-1617]. It was concluded that microtubules are capped with a tubulin-GDP-P(i) subunit at the end of each its 13 protofilaments and that this is responsible for stabilizing microtubules in the growth phase. Also, because microtubules with [(32)P]P(i) were isolated despite the presence of 20 mM P(i), it was concluded that P(i) in terminal tubulin-GDP-P(i) subunits does not exchange with solvent. These observations are inconsistent with our finding that tubulin-GDP-P(i) subunits do not stabilize microtubules and with evidence that the nucleotide, and presumably also P(i), in subunits at microtubule ends exchanges with solvent. We have resolved this discrepancy by finding that during the pulse period the added [(32)P]GTP was almost quantitatively hydrolyzed. The so-formed [(32)P]P(i) labeled the 20 mM P(i) buffer, and this exchanged into tubulin-GDP subunits in the core of the microtubule. Evidence for this was our finding of virtually identical [(32)P]P(i) in microtubules pulsed with [(32)P]GTP with a specific activity that varied 11-fold by using either 100 or 1,100 microM GTP in the reaction. Label uptake was insensitive to the [(32)P]GTP specific activity because in both cases hydrolysis generated 20 mM [(32)P]P(i) with a virtually identical specific activity. Also, approximately 0.4 mol of [(32)P]P(i) /tubulin dimer was found in microtubules when steady-state microtubules in 20 mM P(i) were pulsed with a trace amount of [(32)P]P(i). This stoichiometry is consistent with a 25 mM K(d) previously reported for P(i) binding to tubulin-GDP subunits in microtubules. It is concluded that, under the conditions used for the [(32)P]GTP pulse labeling, (32)P was incorporated into the entire microtubule from [(32)P]P(i) released into the solution, rather than into a tubulin-GDP-P(i) cap, from [(32)P]GTP. Thus, there is no evidence that tubulin-GDP-P(i) subunits accumulate in and stabilize microtubule ends.

Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source.

The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' <--> alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K(d) for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was approximately 9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 microM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K(d) was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.