Yeast secrete ATP in response to glucose, a property with previously unknown functional consequence. In this report, we show that extracellular ATP is a signal for growth of surrounding cells. The ATP signaling behavior was uncovered by finding reduced toxicity of an inducible, dominant-lethal form of alpha tubulin (tub1-828) in cells grown at high, compared to low cell density. Reduced cell death at high cell density resulted because the rate of chromosome loss/cell division was lower (18-fold) in a cultures inoculated with a high density (350,000) compared to a low density (5,000) of cells. The sparing effect of growth at high cell density could be replicated by growing together 3440 cells that express tub1-828, with 2.3 E6 cells that do not express the mutant protein. Toxicity was reduced at high cell density apparently because a secreted signal induces growth, so that the mutant protein is rapidly diluted by synthesis of wild-type α-tubulin. Further, fluorescence-activated cell sorting (FACS) analysis after DNA staining showed that the rate of the G1-G2 transition was faster with cells at high density. ATP replaced the need for high cell density for resistance to tub1-828, and stimulated the transition from G1 to G2 in cells at low density. Cells lacking the enzyme nucleoside diphosphate kinase did not respond to nucleotide stimulation of growth during expression of mutant tubulin, suggesting that NDP kinase has a regulatory role in growth stimulation. This newly discovered quorum sensing response in yeast, mediated by ATP, indicates that yeast decision-making is not entirely autonomous.

Jan Hermans received his PhD in Physical Chemistry from the University of Leiden in 1958 and then had a 2-year postdoctoral fellowship at Cornell with Harold Scheraga. Next, he worked for 2 years at American Viscose Corporation. In 1964, after a year in Genoa with Gianni Rialdi he joined the Department of Biochemistry and Nutrition at the University of North Carolina at Chapel Hill, where he immediately attracted the first of 17 graduate students and 24 postdoctoral trainees he would mentor. Jan's enduring interest was in studies of protein structure and dynamics. While still a graduate student at Leiden, John Kendrew or Max Perutz came to Leiden for a seminar on protein crystallography work. But Jan missed the talk as his advisor told him that the work was uninteresting. He later often remarked that he regretted not having had the opportunity to study crystallography, and his future work made fundamental contributions to the interpretations of electron density maps and macromolecular simulations. Jan's choice of research projects reflected something he repeatedly told associates: you must make something useful. His research contributions confirm that he was truly motivated by this belief: his researches can be grouped into tool development and the use of theoretical and computational approaches to help understand the physics of biochemical processes. His articles are imaginatively resourceful, yet constrained by a high regard for experimental data. An early example is an article coauthored with Harold Scheraga and Nobel laureate Melvin Calvin, observing that, whereas thermal melting temperatures themselves are not directly related to thermodynamic parameters, melting temperature differences induced by shifts in pH, deuteration, and more recently by mutagenesis, are proportional via the overall enthalpy change to changes in the free energy of folding.

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) causes a subset of cases of familial amyotrophic lateral sclerosis (FALS) through structural destabilization of SOD1, which leads to misfolding and aggregation. The non‐heritable (sporadic) nature of most cases of ALS and the late onset of symptoms, even for patients expressing disease‐linked mutant proteins throughout life, suggest a significant role of environmental factor(s) in ALS etiology. We have previously found that SOD1 from human tissue is extensively post‐translationally modified, with the most prevalent modifications being glutathionylation at Cys‐111 and phosphorylation at Thr‐2. We further found that glutathionylation significantly destabilizes SOD1WT dimers, increasing the equilibrium dissociation constant approximately 1,000‐fold, such that its stability is comparable to the A4V FALS‐causative mutation. SOD1A4V is further destabilized by glutathionylation. Using computational structural modeling, we show that glutathionylation has distinct effects on different ALS‐linked SOD1 mutants, which correspond to changes in composition of the dimer interface. Glutathionylation also stabilizes an intermediate misfolded species in which the b‐barrel core of each monomer is “loosened”. These findings suggest a mode by which the cellular environment impacts the propensity of SOD1 to adopt toxic misfolded conformations in 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.

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.

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.

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.

ABSTRACT Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼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 α-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.

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.

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.

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.

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.

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.

The proposal that microtubule dynamic instability results from stabilization of microtubule ends by tubulin-GDP-Pi subunits (where Pi is inorganic phosphate) [Melki et al. (1996) Biochemistry 35, 12038] was based on studies of GTP hydrolysis and microtubule assembly that showed that tubulin-GDP-Pi subunits can transiently accumulate at microtubule ends. There is no direct evidence that GDP-Pi-subunits can stabilize microtubules under conditions where dynamic instability is observed and this has been inferred from the observation that tubulin-GDP-BeFn subunits stabilize microtubules. To test if tubulin-GDP-Pi stabilizes microtubules we sought evidence for a synergism between the effect of Pi and BeFn. We found, however, that Pi antagonizes the effect of BeFn by displacing it from tubulin subunits. The alternate mechanism in which Pi inhibits BeFn stabilization of microtubules by displacing fluoride from beryllium was ruled out from the 9Be and 19F NMR spectra in the presence and absence of Pi. Further evidence that tubulin-GDP-BeFn is not an analogue of tubulin-GDP-Pi and that tubulin-GDP-Pi is not responsible for maintaining the growth phase in microtubules manifesting dynamic instability was provided by our observation that Pi did not decrease the disassembly rate under conditions where tubulin-GDP-Pi subunits are expected to have formed. Results showing that BeFn binds randomly to subunits in microtubules provided evidence that Pi dissociation from the tubulin-GDP-Pi intermediate formed during GTP hydrolysis occurs randomly rather than processively starting at the growing microtubule tip.

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 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 to 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.

The recent discovery that GTP linked to latex beads binds to microtubule ends suggested that nucleotide interactions at this site may play a role in regulating microtubule (MT) dynamics. Evidence for this was sought using DIC microscopy to analyze effects of the free GTP and GDP concentration on the rates of MT elongation and phase transition to rapid shortening (catastrophe, kc). That nucleotide can dissociate and thereby destabilize the plus end by forming nucleotide-free (apotubulin) subunits was indicated by an increase in kc from 0.001 to 0.05 s-1, when the free GTP concentration was reduced from 100 to 0.5 microM, during assembly with 15 microM tubulin--GTP subunits (TuT). That nucleotide can bind to the minus end was indicated by a nearly 5-fold decrease in the rate of elongation when the free GDP concentration was increased from 1.6 to 175 microM, during assembly with a mixture of 36 microM TuT and 54 microM TuD. Further evidence that nucleotide can bind to both ends was provided by the observation that with a mixture of 36 microM TuT and 54 microM TuD, kc was increased from 0.0036 to 0.05 s-1 at the plus end, and from 0.0005 to 0.005 s-1 at the minus end, when the free GDP concentration was increased from 1.6 to 175 microM. Our evidence for destabilization of microtubules by formation of apotubulin and by nucleotide exchange to form terminal TuD subunits suggests that microtubule dynamics can be regulated in cells by an exchange factor that generates apotubulin subunits, or by a GTPase activating protein that forms TuD subunits at microtubule ends.

The standard free energy for hydrolysis of the GTP analogue guanylyl-(a,b)-methylene-diphosphonate (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 GMPCP-microtubules. It was possible to study GMPCPP-microtubules 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 half-time for GMPCPP hydrolysis from > 10 h to approximately 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.