Tubulin Folding Research Articles

Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its β-tubulin binding characterization

Microtubules are composed of polymerized α/β-tubulin heterodimers. Biogenesis of assembly-competent tubulin dimers is a complex multistep process that requires sequential actions of distinct molecular chaperones and cofactors. Tubulin folding cofactor A (TFCA), which captures β-tubulin during the folding pathway, has been identified in many organisms. Here, we report the crystal structure of Arabidopsis thaliana TFC A (KIESEL, KIS), which forms a monomeric three-helix bundle. The functional binding analysis demonstrated that KIS interacts with β-tubulin in plant. Furthermore, mutagenesis studies indicated that the α-helical regions of KIS participate in β-tubulin binding. Unlike the budding yeast TFC A, the two loop regions of KIS are not required for this interaction suggesting a distinct binding mechanism of TFC A to β-tubulin in plants. Structured summary MINT- 7968902, MINT- 7968915, MINT- 7968951, MINT- 7968966: KIS (uniprotkb: O04350) physically interacts (MI: 0915) with Tub9 (uniprotkb: P29517) by anti tag coimmunoprecipitation (MI: 0007) MINT- 7968928: KIS (uniprotkb: O04350) and Tub9 (uniprotkb: P29517) physically interact (MI: 0915) by bimolecular fluorescence complementation (MI: 0809)

Disease-associated mutations in TUBA1A result in a spectrum of defects in the tubulin folding and heterodimer assembly pathway

Malformations of cortical development are characteristic of a plethora of diseases that includes polymicrogyria, periventricular and subcortical heterotopia and lissencephaly. Mutations in TUBA1A and TUBB2B, each a member of the multigene families that encode alpha- and beta-tubulins, have recently been implicated in these diseases. Here we examine the defects that result from nine disease-causing mutations (I188L, I238V, P263T, L286F, V303G, L397P, R402C, 402H, S419L) in TUBA1A. We show that the expression of all the mutant proteins in vitro results in the generation of tubulin heterodimers in varying yield and that these can co-polymerize with microtubules in vitro. We identify several kinds of defects that result from these mutations. Among these are various defects in the chaperone-dependent pathway leading to de novo tubulin heterodimer formation. These include a defective interaction with the chaperone prefoldin, a reduced efficiency in the generation of productive folding intermediates as a result of inefficient interaction with the cytosolic chaperonin, CCT, and, in several cases, a failure to stably interact with TBCB, one of five tubulin-specific chaperones that act downstream of CCT in the tubulin heterodimer assembly pathway. Other defects include structural instability in vitro, diminished stability in vivo, a compromised ability to co-assemble with microtubules in vivo and a suppression of microtubule growth rate in the neurites (but not the soma) of cultured neurons. Our data are consistent with the notion that some mutations in TUBA1A result in tubulin deficit, whereas others reflect compromised interactions with one or more MAPs that are essential to proper neuronal migration.

Equivalent Mutations in the Eight Subunits of the Chaperonin CCT Produce Dramatically Different Cellular and Gene Expression Phenotypes

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.

Tubulin binding cofactor C (TBCC) suppresses tumor growth and enhances chemosensitivity in human breast cancer cells

BackgroundMicrotubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC), a crucial protein for the proper folding of α and β tubulins into polymerization-competent tubulin heterodimers.MethodsWe developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells.ResultsTBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to antimicrotubule agents both in vitro and in xenografts.ConclusionThese results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterisation of resistant phenotypes.

A long hangover from party drugs: Residual proteomic changes in the hippocampus of rats 8 weeks after γ-hydroxybutyrate (GHB), 3,4-methylenedioxymethamphetamine (MDMA) or their combination

3,4-Methylenedioxymethamphetamine (MDMA) and γ-hydroxybutyrate (GHB) are popular party drugs that are used for their euphoric and prosocial effects, and sometimes in combination. Both drugs increase markers of oxidative stress in the hippocampus and can cause lasting impairments in hippocampal-dependent forms of memory. To gain further information on the biochemical mechanisms underlying these effects, the current study examined residual changes in hippocampal protein expression measured 8 weeks after chronic administration of GHB (500mg/kg), MDMA (5mg/kg) or their combination (GHB/MDMA). The drugs were administered once a day for 10 days in an environment with an elevated ambient temperature of 28°C. Results showed significant changes in protein expression, relative to controls, in all three groups: MDMA and GHB given alone caused residual changes in 8 and 5 proteins respectively, while the GHB/MDMA combination significantly changed 6 proteins. The altered proteins had roles in neuroplasticity, neuroprotection, intracellular signalling and cytoskeletal function. The largest change (−4.3-fold) was seen in the MDMA group with the protein C-crk: a protein implicated in learning-related neuroplasticity. The second largest change (3.0-fold) was seen in the GHB group in Glutathione-S-transferase (GST), a protein that protects against oxidative stress. Two cytoskeletal proteins (Tubulin Folding Cofactor B and Tropomyosin-alpha-3 chain) and one plasticity related protein (Neuronal Pentraxin-1 NP1) were similarly changed in both the MDMA and the GHB groups, while two intracellular signalling proteins (alpha-soluble NSF-attachment protein and subunits of the V-type proton ATPase) were changed in both the MDMA/GHB and the MDMA groups. These results provide some insight into the molecular pathways possibly underlying the lasting cognitive deficits arising from GHB and/or MDMA use.

Comparative Functional Genomic Analysis Identifies Distinct and Overlapping Sets of Genes Required for Resistance to Monomethylarsonous Acid (MMAIII) and Arsenite (AsIII) in Yeast

Arsenic is a human toxin and carcinogen commonly found as a contaminant in drinking water. Arsenite (As(III)) is the most toxic inorganic form, but recent evidence indicates that the metabolite monomethylarsonous acid (MMA(III)) is even more toxic. We have used a chemical genomics approach to identify the genes that modulate the cellular toxicity of MMA(III) and As(III) in the yeast Saccharomyces cerevisiae. Functional profiling using homozygous deletion mutants provided evidence of the requirement of highly conserved biological processes in the response against both arsenicals including tubulin folding, DNA double-strand break repair, and chromatin modification. At the equitoxic doses of 150 microM MMA(III) and 300 microM As(III), genes related to glutathione metabolism were essential only for resistance to the former, suggesting a higher potency of MMA(III) to disrupt glutathione metabolism than As(III). Treatments with MMA(III) induced a significant increase in glutathione levels in the wild-type strain, which correlated to the requirement of genes from the sulfur and methionine metabolic pathways and was consistent with the induction of oxidative stress. Based on the relative sensitivity of deletion strains deficient in GSH metabolism and tubulin folding processes, oxidative stress appeared to be the primary mechanism of MMA(III) toxicity whereas secondary to tubulin disruption in the case of As(III). Many of the identified yeast genes have orthologs in humans that could potentially modulate arsenic toxicity in a similar manner as their yeast counterparts.

NMR Solution Structure of a Tubulin folding cofactor B obtained from Arabidopsis thaliana: Northeast Structural Genomics Consortium target AR3436A

NMR Solution Structure of a Tubulin folding cofactor B obtained from Arabidopsis thaliana: Northeast Structural Genomics Consortium target AR3436A

ASQ2 Encodes a TBCC-like Protein Required for Mother-Daughter Centriole Linkage and Mitotic Spindle Orientation

An intriguing feature of centrioles is that these highly complicated microtubule-based structures duplicate once per cell cycle, affording the cell precise control over their number. Each cell contains exactly two centrioles, linked together as a pair, one of which is a mother centriole formed in a previous cell cycle and the other of which is a daughter centriole whose assembly is templated by the mother. Neither the molecular basis nor the functional role of mother-daughter centriole linkage is understood. We have identified a mutant, asq2, with defects in centriole linkage. asq2 mutant cells have variable numbers of centrioles and centriole positioning defects. Here, we show that ASQ2 encodes the conserved protein Tbccd1, a member of a protein family including a tubulin folding cochaperone and the retinitis pigmentosa protein RP2, involved in tubulin quality control during ciliogenesis. We characterize mitosis in asq2 cells and show that the majority of cells establish a bipolar spindle but have defects in spindle orientation. Few asq2 cells have centrioles at both poles, and these cells have properly positioned spindles, indicating that centrioles at the poles might be important for spindle orientation. The defects in centriole number control, centriole positioning, and spindle orientation appear to arise from perturbation of centriole linkage mediated by Tbccd1/Asq2p.

Prefoldins 3 and 5 Play an Essential Role in Arabidopsis Tolerance to Salt Stress

During the last years, our understanding of the mechanisms that control plant response to salt stress has been steadily progressing. Pharmacological studies have allowed the suggestion that the cytoskeleton may be involved in regulating such a response. Nevertheless, genetic evidence establishing that the cytoskeleton has a role in plant tolerance to salt stress has not been reported yet. Here, we have characterized Arabidopsis T-DNA mutants for genes encoding proteins orthologous to prefoldin (PFD) subunits 3 and 5 from yeast and mammals. In these organisms, PFD subunits, also known as Genes Involved in Microtubule biogenesis (GIM), form a heterohexameric PFD complex implicated in tubulin and actin folding. We show that, indeed, PFD3 and PFD5 can substitute for the loss of their yeast orthologs, as they are able to complement yeast gim2Delta and gim5Delta mutants, respectively. Our results indicate that pfd3 and pfd5 mutants have reduced levels of alpha- and beta-tubulin compared to the wild-type plants when growing under both control and salt-stress conditions. In addition, pfd3 and pfd5 mutants display alterations in their developmental patterns and microtubule organization, and, more importantly, are hypersensitive to high concentrations of NaCl but not of LiCl or mannitol. These results demonstrate that the cytoskeleton plays an essential role in plant tolerance to salt stress.

Post-chaperonin tubulin folding pathway

Post-chaperonin tubulin folding pathway

Post Chaperonin tubulin folding pathway

Post Chaperonin tubulin folding pathway

Minimal Plus-end Tracking Unit of the Cytoplasmic Linker Protein CLIP-170

Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (+TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 +TIP behavior is unknown, and many potential models have been presented. Here, by separating the two CLIP-170 CAP-Gly domains and their adjacent serine-rich regions into fragments of varied size, we have characterized the minimal plus-end tracking unit of CLIP-170 in vivo. Each CLIP-170 fragment was also characterized for its tubulin polymerization activity in vitro. We found that the two CAP-Gly domains have different activities, whereas CAP-Gly-1 appears incompetent to mediate either +TIP behavior or MT nucleation, a CLIP-170 fragment consisting of the second CAP-Gly domain and its adjacent serine-rich region can both track MT plus ends in vivo and induce tubulin polymerization in vitro. These observations complement recent work on CLIP-170 fragments, demonstrate that CAP-Gly motifs do not require dimerization for +TIP and polymerization-promoting activities, and provide insight into CLIP-170 function and mechanism.

Characterization of the Group II Chaperonin TriC derived from Human Cervical Adenocarcinoma (HeLa) Cells

Molecular chaperones are key elements in protein folding pathways, and there are numerous proteins which are unable to fold in the absence of one or more molecular chaperones. Eukaryotic chaperones can be broadly separated into two families: chaperones, which sequester and protect unfolded proteins, but do not facilitate folding, and chaperonins, which require ATP, and actively fold nacent or misfolded proteins. The mammalian chaperonin TRiC, in addition to being required for actin and tubulin folding, binds and refold several disease causing proteins in vitro, including those associated with Alzheimer's disease, Von Hippel Landau tumor, and Huntington's disease. This refolding activity has revealed TRiC as a possible therapeutic agent for the treatment and prevention of aggregation diseases. We have purified TriC from cervical adenocarcinoma cells. To assess the properties of Human TriC, we are characterizing its interactions of with human γ Crystallins. Crystallins are a family of structural proteins found in the lens of the human eye, and aggregation of these proteins is thought to be the cause of cataract. Methanococcus marapaludis chaperonin Mm-Cpn, a homolog of human TriC, has been shown to both suppress aggregation of Human γ Crystallins, and refold the Crystallins to a native-like conformation. Suppression of γ Crystallin aggregation is being investigated using UV/Vis spectroscopy, and the ability of TriC to restore unfolded γ Crystallin to a native fold investigated using size exclusion chromatography. In collaboration with fellow members of the Center for Protein Folding Machinery, we are investigating the possibility of visualizing the crystallin substrate in the chaperonin/substrate complex by Cryo-EM.Supported by an NIH Roadmap Award to the Center for Protein Folding Machinery (http://ncmi.bcm.tmc.edu/nanomedicine).

Gigaxonin controls vimentin organization through a tubulin chaperone-independent pathway

Gigaxonin mutations cause the fatal human neurodegenerative disorder giant axonal neuropathy (GAN). Broad deterioration of the nervous system in GAN patients is accompanied by massive disorganization of intermediate filaments (IFs) both in neurons and many non-neuronal cells. With newly developed antibodies, gigaxonin is now shown to be expressed at extremely low levels throughout the nervous system. In lymphoblast cell lines derived from severe and mild forms of GAN, mutations in gigaxonin are shown to yield highly unstable proteins, thereby permitting a rapid diagnostic test for the spectrum of GAN mutations as an alternative to invasive nerve biopsy or systematic sequencing of the GAN gene. Gigaxonin has been proposed as a substrate adaptor for an E3 ubiquitin ligase, which affects proteasome-dependent degradation of microtubule-related proteins including MAP1B, MAP8 and the tubulin folding chaperone TBCB. We demonstrate that, unlike its counterpart TBCE, TBCB only moderately destabilizes microtubules. Neither TBCB abundance nor microtubule organization or densities are altered in GAN mutant fibroblasts, thus demonstrating that altered TBCB levels are not primary determinants of IF disorganization in GAN. Characteristic GAN mutant-induced ovoid aggregates of vimentin are not produced in normal fibroblasts after disrupting microtubule assembly, either by TBCE overexpression or depolymerizing drugs. Thus, IF disorganization in GAN fibroblasts is independent of TBCB and microtubule loss and must be regulated by a yet unidentified mechanism.

Translation termination and protein folding pathway genes are not correlated in gastric cancer

The eukaryotic release factor 3 (eRF3) has been shown to affect both tubulin and actin cytoskeleton, suggesting a role in cytoskeleton assembly, mitotic spindle formation and chromosome segregation. Also, direct interactions between eRF3 and subunits of the cytosolic chaperonin CCT have been described. Moreover, both eRF3a and CCT subunits have been described to be up-regulated in cancer tissues. Our aim was to evaluate the hypothesis that eRF3 expression levels are correlated with the expression of genes encoding proteins involved in the tubulin folding pathways. Relative expression levels of eRF1, eRF3a/GSPT1, PFDN4, CCT2, CCT4, and TBCA genes in tumour samples relative to their adjacent normal tissues were investigated using real time-polymerase chain reaction in 20 gastric cancer patients. The expression levels of eRF3a/GSPT1 were not correlated with the expression levels of the other genes studied. However, significant correlations were detected between the other genes, both within intestinal and diffuse type tumours. eRF3a/GSPT1 expression at the mRNA level is independent from both cell translation rates and from the expression of the genes involved in tubulin-folding pathways. The differences in the patterns of expression of the genes studied support the hypothesis of genetically independent pathways in the origin of intestinal and diffuse type gastric tumours.

Cofactor D Functions as a Centrosomal Protein and Is Required for the Recruitment of the γ-Tubulin Ring Complex at Centrosomes and Organization of the Mitotic Spindle

Microtubules are highly dynamic structures, composed of alpha/beta-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the full-length protein or either of two centrosome localization domains leads to the loss of anchoring of the gamma-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of alpha-or beta-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.

Tubulin Folding Pathways: Implication in the Regulation of Microtubule Dynamics

As microtubules are essential in many cell functions, they have been used as a target of a variety of anticancer drugs that are grouped as stabilizing (taxanes) and destabilizing (vinca-alkaloids, colchicinoids) microtubule agents. It appears clearly now that the dynamic behaviour more than modifications of microtubule mass are altered by antitubulin agents in the range of serum concentrations obtained after administration in humans. While the role of microtubule associated proteins in the regulation of microtubule dynamics has been extensively studied, there is a growing body of data suggesting that tubulin folding could also play an important role in microtubule dynamics. We review the current knowledge regarding tubulin folding pathways, their relation to disease, and their possible influence on microtubule dynamics.

Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans

The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in α-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 ( pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.

Hypoparathyroidism, Retardation, and Dysmorphism Syndrome: Impaired Early Growth and Increased Susceptibility to Severe Infections Due to Hyposplenism and Impaired Polymorphonuclear Cell Functions

Hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome is the first reported disease caused by a defect in the tubulin folding and assembly pathway. We aimed to summarize our experience with a cohort of patients with HRD, analyze their growth, and evaluate patients' polymorphonuclear cell (PMN) functions. The records of 22 HRD patients in a single medical center were reviewed. Growth during infancy and early childhood were analyzed by the Infancy-Childhood-Puberty (ICP) growth model. PMN functions were compared with healthy controls. Twelve patients died and many hospitalizations due to infections and convulsions were recorded. Growth measurements, expressed as weight and height SD scores in boys at a mean age of 4 y were -13.1+/-3.8 and -8.7+/-1 and -16.6+/-4.4 and -9.5+/-2.4, respectively, in girls at a mean age of 6.4 y. Chemotactic migration, random migration, and phagocytosis of PMN from HRD patients were significantly lower than that of PMN from healthy controls. No significant differences were found in superoxide production of PMN from patients compared with controls. Functional hyposplenism has been demonstrated in most of the studied patients. The defect in the tubulin folding and assembly pathway, previously described in HRD, has grave consequences on growth and PMN functions.

Distinct tubulin genes are differentially expressed during barley grain development

Tubulins, as major components involved in the organization of microtubules, play an important role in plant development. We describe here the expression profiles of all known alpha-tubulin (TUA), beta-tubulin (TUB) and gamma-tubulin (TUG) genes of barley (Hordeum vulgare), involving eight newly identified TUB sequences, five established TUA genes and one TUG gene. Macroarray and Northern blot-based expression patterns in the pericarp, endosperm and embryo were obtained over the course of the development of the grain between anthesis and maturation. These revealed that the various tubulin genes differed in their levels of expression, and to some extent were tissue specific. Two expression peaks were detected in the developing endosperm. The first and more prominent peak, at 2 days after flowering, included expression of almost all the tubulin genes. These tubulins are thought to be involved in mitoses during the formation of the syncytial endosperm. The second, less pronounced but more extended, peak included only some of the tubulin genes (HvTUA3, HvTUB1 and HvTUG) and might be associated with the cell wall organization in aleurone and starchy endosperm. The HvTUA5 gene is expressed only in embryo of the developing grain and may be associated with shoot establishment. The expression profiles of the tubulin folding cofactors HvTFC A and HvTFC B as well as small G-protein HvArl2 genes were almost perfectly correlated with the global levels of tubulin mRNA, implying that they have a role in the control of the polymerization of alpha/beta-tubulin heterodimers.