Tubulin Isotypes Research Articles

The Tubulin Code in Mitosis and Cancer.

The “tubulin code” combines different α/β-tubulin isotypes with several post-translational modifications (PTMs) to generate microtubule diversity in cells. During cell division, specific microtubule populations in the mitotic spindle are differentially modified, but only recently, the functional significance of the tubulin code, with particular emphasis on the role specified by tubulin PTMs, started to be elucidated. This is the case of α-tubulin detyrosination, which was shown to guide chromosomes during congression to the metaphase plate and allow the discrimination of mitotic errors, whose correction is required to prevent chromosomal instability—a hallmark of human cancers implicated in tumor evolution and metastasis. Although alterations in the expression of certain tubulin isotypes and associated PTMs have been reported in human cancers, it remains unclear whether and how the tubulin code has any functional implications for cancer cell properties. Here, we review the role of the tubulin code in chromosome segregation during mitosis and how it impacts cancer cell properties. In this context, we discuss the existence of an emerging “cancer tubulin code” and the respective implications for diagnostic, prognostic and therapeutic purposes.

A new method for protein characterization and classification using geometrical features for 3D face analysis: An example of tubulin structures.

This article reports on the results of research aimed to translate biometric 3D face recognition concepts and algorithms into the field of protein biophysics in order to precisely and rapidly classify morphological features of protein surfaces. Both human faces and protein surfaces are free-forms and some descriptors used in differential geometry can be used to describe them applying the principles of feature extraction developed for computer vision and pattern recognition. The first part of this study focused on building the protein dataset using a simulation tool and performing feature extraction using novel geometrical descriptors. The second part tested the method on two examples, first involved a classification of tubulin isotypes and the second compared tubulin with the FtsZ protein, which is its bacterial analog. An additional test involved several unrelated proteins. Different classification methodologies have been used: a classic approach with a support vector machine (SVM) classifier and an unsupervised learning with a k-means approach. The best result was obtained with SVM and the radial basis function kernel. The results are significant and competitive with the state-of-the-art protein classification methods. This leads to a new methodological direction in protein structure analysis.

Tubulin post-translational modifications control neuronal development and functions.

Microtubules (MTs) are an essential component of the neuronal cytoskeleton; they are involved in various aspects of neuron development, maintenance, and functions including polarization, synaptic plasticity, and transport. Neuronal MTs are highly heterogeneous due to the presence of multiple tubulin isotypes and extensive post‐translational modifications (PTMs). These PTMs—most notably detyrosination, acetylation, and polyglutamylation—have emerged as important regulators of the neuronal microtubule cytoskeleton. With this review, we summarize what is currently known about the impact of tubulin PTMs on microtubule dynamics, neuronal differentiation, plasticity, and transport as well as on brain function in normal and pathological conditions, in particular during neuro‐degeneration. The main therapeutic approaches to neuro‐diseases based on the modulation of tubulin PTMs are also summarized. Overall, the review indicates how tubulin PTMs can generate a large number of functionally specialized microtubule sub‐networks, each of which is crucial to specific neuronal features.

Intrinsic and Extrinsic Factors Affecting Microtubule Dynamics in Normal and Cancer Cells.

Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule–MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the consequences of such a phenomenon, including drug resistance development.

A Confirmation for the Positive Electric Charge of Bio-Molecular Motors through Utilizing a Novel Nano-Technology Approach In Vitro.

Molecular motors are microtubule-based proteins which contribute to many cell functions, such as intracellular transportation and cell division. The details of the nature of the mutual interactions between motors and microtubules still needs to be extensively explored. However, electrostatic interaction is known as one of the key factors making motor-microtubule association possible. The association rate of molecular motors to microtubules is a way to observe and evaluate the charge of the bio-motors in vivo. Growing evidence indicates that microtubules with distinct structural compositions in terms of beta tubulin isotypes carry different charges. Therefore, the electrostatic-driven association rate of motors–microtubules, which is a base for identifying the charge of motors, can be more likely influenced. Here, we present a novel method to experimentally confirm the charge of molecular motors in vitro. The offered nanotechnology-based approach can validate the charge of motors in the absence of any cellular components through the observation and analysis of the changes that biomolecular motors can cause on the dynamic of charged microspheres inside a uniform electric field produced by a microscope slide-based nanocapacitor. This new in vitro experimental method is significant as it minimizes the intracellular factors that may interfere the electric charge that molecular motors carry.

High TUBB2A expression in childhood T‐ALL is correlated with the clinical outcome

AbstractIntroductionMicrotubules are polymers that perform functions such as mitosis, intracellular transport, cell morphology, and ciliary and flagellar motility. Since microtubules are taking active part in cell division, they are among direct targets of several antimitotic drugs.MethodsExpression levels of tubulin isotypes were analyzed in microarray data of childhood diagnostic T‐ALL samples (n = 31) and healthy thymocytes (n = 7). Findings were validated with qPCR in separate T‐ALL cohort (n = 48), and clinical correlation analyses were performed. TUBB2A's effects were tested with siRNA‐mediated knockdown in MOLT4 cell line, and apoptosis assay was carried out at 24, 48, and 72 hours time points.ResultsIn microarray data, TUBB2A was found to be the only differentially expressed tubulin isotype (adj. P value = .01), which was validated by qPCR (P = .02). Samples representing differentiation stages of T cell showed an increasing trend of TUBB2A toward mature T‐cell stage. TUBB2A expression was significantly higher in high‐risk group patients (P = .026) and in a group with WBC counts >100 (×109cells/L) (P = .029). High TUBB2A was also found to be a predictor of shorter OS (P = .029) and RFS (P = .042).ConclusionAberrant expression of TUBB isotypes can affect the balance of microtubules or microtubule‐associated proteins, which might lead to drug resistance/relapse. Contribution of cytoskeleton proteins to drug resistance needs further investigation, and understanding aberrant expression and mode of action of microtubules will improve therapy strategies.

Primary myeloid cell proteomics and transcriptomics: importance of β-tubulin isotypes for osteoclast function.

Among hematopoietic cells, osteoclasts (OCs) and immature dendritic cells (DCs) are closely related myeloid cells with distinct functions: OCs participate skeleton maintenance while DCs sample the environment for foreign antigens. Such specificities rely on profound modifications of gene and protein expression during OC and DC differentiation. We provide global proteomic and transcriptomic analyses of primary mouse OCs and DCs, based on original stable isotope labeling with amino acids in cell culture (SILAC) and RNAseq data. We established specific signatures for OCs and DCs, including genes and proteins of unknown functions. In particular, we showed that OCs and DCs have the same α- and β-tubulin isotype repertoire but that OCs express much more of the β tubulin isotype Tubb6 (also known as TBB6). In both mouse and human OCs, we demonstrate that elevated expression of Tubb6 in OCs is necessary for correct podosomes organization and thus for the structure of the sealing zone, which sustains the bone resorption apparatus. Hence, lowering Tubb6 expression hinders OC resorption activity. Overall, we highlight here potential new regulators of OC and DC biology, and illustrate the functional importance of the tubulin isotype repertoire in the biology of differentiated cells.

Identification of β tubulin IVb as a pattern recognition receptor with opsonic activity

Previous studies have shown that tubulins play important role in immune responses of both plants and animals, but no experiments have been performed to study the mode of action of tubulins in immune defense. In addition, there is little convincing experimental evidence of functional commitment for specific tubulin isotypes in animals. In the present, we showed that expression of β-tubulin IVb gene was affected by both LPS and LTA, hinting its involvement in anti-infectious response. We also showed that recombinant zebrafish β-tubulin IVb not only interacted with LPS and LTA as well as Gram-negative and -positive bacteria but also agglutinated both Gram-negative and -positive bacteria in a Ca2+-dependent fashion. Interestingly, recombinant β-tubulin IVb could enhance the phagocytosis of bacteria by macrophages. Moreover, we demonstrated that β-tubulin IVb was present extracellularly in the serum of zebrafish and mouse. Collectively, these suggest that β-tubulin IVb may be physiologically involved in the systematic immunity of host via acting as a pattern recognition receptor and an opsonin. This also provides a new angle to understand the roles of β-tubulin IVb.

Alpha- and beta-tubulin isotypes are differentially expressed during brain development.

Alpha- and beta-tubulin dimers polymerize into protofilaments that associate laterally to constitute a hollow tube, the microtubule. A dynamic network of interlinking filaments forms the microtubule cytoskeleton, which maintains the structure of cells and is key to various cellular processes including cell division, cell migration, and intracellular transport. Individual microtubules have an identity that depends on the differential integration of specific alpha- and beta-tubulin isotypes and is further specified by a variety of posttranslational modifications (PTMs). It is barely understood to which extent neighboring microtubules differ in their tubulin composition or whether specific tubulin isotypes cluster along the polymer. Furthermore, our knowledge about the spatio-temporal expression patterns of tubulin isotypes is limited, not at least due to the lack of antibodies or antibody cross-reactivities. Here, we asked which alpha- and beta-tubulin mRNAs and proteins are expressed in developing hippocampal neuron cultures and ex vivo brain tissue lysates. Using heterologous expression of GFP-tubulin fusion proteins, we systematically tested antibody-specificities against various tubulin isotypes. Our data provide quantitative information about tubulin expression levels in the mouse brain and classify tubulin isotypes during pre- and postnatal development.

Exploring the interaction of Peloruside-A with drug resistant αβII and αβIII tubulin isotypes in human ovarian carcinoma using a molecular modeling approach

Microtubules (MTs) play an essential role in mitosis; hence they are identified as potential targets to design novel anti-mitotic agents. MT’s are composed of α/β-tubulin isotypes that are associated with differential drug-resistant effects against MT-targeting agents. Peloruside-A (PLA) is a potent anti-mitotic agent showing excellent activity against taxol-resistant carcinoma. PLA alters MT dynamics by binding to the ‘non-taxoid’ site of β-tubulin. The abundance of βII and βIII tubulin isotypes in human ovarian carcinoma affects the efficacy of PLA. Nevertheless, the mechanism of PLA resistance due to βII and βIII tubulin isotype is not well understood. Therefore, we investigated the interactions of PLA with αβIIa, αβIIb, and αβIII tubulin isotypes which are predominantly expressed in the human ovarian carcinoma, using a molecular modeling approach. A sequence analysis study shows that the βIII isotype has seven residue variations at the ‘non-taxoid’ site compared to the βIIa and βIIb isotypes. Molecular docking and molecular dynamics simulation revealed that residue variation at the ‘non-taxoid’ site of βIII isotype affect PLA binding. Furthermore, binding energy calculations showed that αβIIa has the highest binding towards PLA, whereas αβIIb and αβIII isotypes shows weaker associations with PLA. Our computational study provides valuable structural and energetic information to increase understanding into the origin of PLA resistance in human ovarian carcinoma and could be helpful to develop potential PLA analogs against specific β-tubulin isotypes expressed in cancer cells. Communicated by Ramaswamy H. Sarma

The tubulin code and its role in controlling microtubule properties and functions.

Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the 'tubulin code'. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.

In silico Investigations of the Mode of Action of Novel Colchicine Derivatives Targeting β-Tubulin Isotypes: A Search for a Selective and Specific β-III Tubulin Ligand.

The cardinal role of microtubules in cell mitosis makes them interesting drug targets for many pharmacological treatments, including those against cancer. Moreover, different expression patterns between cell types for several tubulin isotypes represent a great opportunity to improve the selectivity and specificity of the employed drugs and to design novel compounds with higher activity only on cells of interest. In this context, tubulin isotype βIII represents an excellent target for anti-tumoral therapies since it is overexpressed in most cancer cells and correlated with drug resistance. Colchicine is a well-known antimitotic agent, which is able to bind the tubulin dimer and to halt the mitotic process. However, it shows high toxicity also on normal cells and it is not specific for isotype βIII. In this context, the search for colchicine derivatives is a matter of great importance in cancer research. In this study, homology modeling techniques, molecular docking, and molecular dynamics simulations have been employed to characterize the interaction between 55 new promising colchicine derivatives and tubulin isotype βIII. These compounds were screened and ranked based on their binding affinity and conformational stability in the colchicine binding site of tubulin βIII. Results from this study point the attention on an amide of 4-chlorine thiocolchicine. This colchicine-derivative is characterized by a unique mode of interaction with tubulin, compared to all other compounds considered, which is primarily characterized by the involvement of the α-T5 loop, a key player in the colchicine binding site. Information provided by the present study may be particularly important in the rational design of colchicine-derivatives targeting drug resistant cancer phenotypes.

Synthesis, biological evaluation and molecular docking studies of new amides of 4-chlorothiocolchicine as anticancer agents.

Colchicine belongs to a large group of microtubule polymerization inhibitors. Although the anti-cancer activity of colchicine and its derivatives has been established, none of them has found commercial application in cancer treatment due to side effects. Therefore, we designed and synthesized a series of six triple-modified 4-chlorothiocolchicine analogues with amide moieties and one urea derivative. These novel derivatives were tested against several different cancer cell lines (A549, MCF-7, LoVo, LoVo/DX) and primary acute lymphoblastic leukemia (ALL) cells and they showed activity in the nanomolar range. The obtained IC50 values for novel derivatives were lower than those obtained for unmodified colchicine and common anticancer drugs such as doxorubicin and cisplatin. Further studies of colchicine and selected analogues were undertaken to indicate that they induced apoptotic cell death in ALL-5 cells. We also performed in silico studies to predict binding modes of the 4-chlorothiocolchicine derivatives to different β tubulin isotypes. The results indicate that select triple-modified 4-chlorothiocolchicine derivatives represent highly promising novel cancer chemotherapeutics.

Tuba8 Drives Differentiation of Cortical Radial Glia into Apical Intermediate Progenitors by Tuning Modifications of Tubulin C Termini.

Most adult neurons and glia originate from radial glial progenitors (RGs), a type of stem cell typically extending from the apical to the basal side of the developing cortex. Precise regulation of the choice between RG self-renewal and differentiation is critical for normal development, but the mechanisms underlying this transition remain elusive. We show that the non-canonical tubulin Tuba8, transiently expressed in cortical progenitors, drives differentiation of RGs into apical intermediate progenitors, a more restricted progenitor type lacking attachment to the basal lamina. This effect depends on the unique C-terminal sequence of Tuba8 that antagonizes tubulin tyrosination and Δ2 cleavage, two post-translational modifications (PTMs) essential for RG fiber maintenance and the switch between direct and indirect neurogenesis and ultimately distinct neuronal lineage outcomes. Our work uncovers aninstructive role of a developmentally regulated tubulin isotype in progenitor differentiation and provides new insights into biological functions of the cellular tubulin PTM "code."

An Atomic Level Interactions of Phosphorylated Tau Repeat with Microtubule using Molecular Modeling Approach

Tau is a microtubule-associated protein abundantly expressed in brain and neuronal cells. Microtubules (MT) are madeup of αβ tubulin heterodimeric subunits. Seven α-tubulin and nine β-tubulin isotypes are reported in humans till date, of which βIII tubulin isotypes are predominantly expressed in the neuronal cells and brain. The C-terminal repeat regions composed of R1, R2, R3 and R4 binds to stabilizes the axonal MT. In several neurodegenerative diseases, tau detaches from MT to form insoluble aggregates leading to tauopathy. Phosphorylation of tau is important for the regulating its structure and function. However, hyperphosphorylation of tau is causes its detachment from the MT. The molecular mechanism which triggers detachment of hyperphosphorylated tau from the MT remains elusive. Therefore, we studied interactions of phosphorylated tau repeat region R2 (TauR2) with neuronal specific βIII tubulin isotypes. Our MD simulation results show a single and double phosphorylation in the TauR2 does not affect the MT binding. However, hyperphosphorylation of TauR2 causes its detachment from the MT. The negatively charged C-terminal tail of the βIII tubulin repels negatively charged phosphorylated TauR2, hence causes the detachment. Also, phosphorylated TauR2 shows poor binding affinity towards neuronal specific MT composed of βIII tubulin isotypes. Our strategy can be potentially used to understand detachment of tau from the MTs composed of different β-tubulin isotypes expressed in specific neurodegenerative disorders. We hope that knowledge of precise molecular interactions between phosphorylated tau repeat and neuronal specific MT will pave the way for developing effective treatments against tau related disorders.

Genetic polymorphism of p- Tubulin isotype 1 gene conferring benzimidazole resistance in Haemonchus contortus in sheep of Andhra Pradesh

Genetic polymorphism of p- Tubulin isotype 1 gene conferring benzimidazole resistance in Haemonchus contortus in sheep of Andhra Pradesh

Tubulin mutations in brain development disorders: Why haploinsufficiency does not explain TUBA1A tubulinopathies.

The neuronal cytoskeleton performs incredible feats during nervous system development. Extension of neuronal processes, migration, and synapse formation rely on the proper regulation of microtubules. Mutations that disrupt the primary α-tubulin expressed during brain development, TUBA1A, are associated with a spectrum of human brain malformations. One model posits that TUBA1A mutations lead to a reduction in tubulin subunits available for microtubule polymerization, which represents a haploinsufficiency mechanism. We propose an alternative model for the majority of tubulinopathy mutations, in which the mutant tubulin polymerizes into the microtubule lattice to dominantly "poison" microtubule function. Nine distinct α-tubulin and ten β-tubulin genes have been identified in the human genome. These genes encode similar tubulin proteins, called isotypes. Multiple tubulin isotypes may partially compensate for heterozygous deletion of a tubulin gene, but may not overcome the disruption caused by missense mutations that dominantly alter microtubule function. Here, we describe disorders attributed to haploinsufficiency versus dominant negative mechanisms to demonstrate the hallmark features of each disorder. We summarize literature on mouse models that represent both knockout and point mutants in tubulin genes, with an emphasis on how these mutations might provide insight into the nature of tubulinopathy patient mutations. Finally, we present data from a panel of TUBA1A tubulinopathy mutations generated in yeast α-tubulin that demonstrate that α-tubulin mutants can incorporate into the microtubule network and support viability of yeast growth. This perspective on tubulinopathy mutations draws on previous studies and additional data to provide a fresh perspective on how TUBA1A mutations disrupt neurodevelopment.

Structure, Thermodynamics, and Kinetics of Plinabulin Binding to Two Tubulin Isotypes

Summary αβ-Tubulin is a validated target for anticancer drug discovery, and molecules binding to this protein are used to treat several types of tumors. Here, we report on a combined X-ray crystallography and molecular dynamics approach to study drug binding within the colchicine site of αβ-tubulin, focusing on plinabulin, an agent currently in phase 3 clinical testing for the treatment of cancer and chemotherapy-induced neutropenia. We found that plinabulin is more persistently bound to the colchicine site of βII- compared to βIII-tubulin, allowing for a prediction of isotype-expression-dependent drug sensitivity. Additionally, computational residence time and exit paths from the βII-tubulin were compared between plinabulin and two other compounds, colchicine and combretastatin-A4. The former displayed the highest residence time, followed by plinabulin and then distantly by combretastatin-A4. Our combined experimental and computational protocol could help to investigate anti-tubulin drugs, improving our understanding of their mechanism of action, residence time, and tubulin isotype selectivity.

Lower Tubulin Expression in Glioblastoma Stem Cells Attenuates Efficacy of Microtubule-Targeting Agents.

Sensitivity to microtubule-targeting agents (MTAs) varies among cancers and predicting the response of individual cancer patients to MTAs remains challenging. As microtubules possess vast molecular heterogeneity generated by tubulin isotypes and their post-translational modifications, we questioned whether this heterogeneity can impact MTA sensitivity. We investigated microtubule heterogeneity in 15 glioblastoma cell lines and measured sensitivity of orthogonal MTAs using a per-division growth rate inhibition method that corrects for the confounding effects of variable cell proliferation rates. We found that the tubulin profile is unique for each glioblastoma cell line and that the total α- and β-tubulin levels impact on MTA sensitivity. The baseline levels of α- and β-tubulin were up to 20% lower in cells that were not effectively killed by MTAs. We report that lower α/β-tubulin expression is associated with lack of cell differentiation and increased expression of stemness markers. The dedifferentiated stem-like cells with low α/β-tubulin levels survive MTAs treatment via reversible nonmutational dormancy. Our findings provide novel insights into the relationships between microtubules and MTAs and lay a foundation for better understanding of the sensitivity of cancer cells to MTAs.

Differential binding affinity of tau repeat region R2 with neuronal-specific \u03b2-tubulin isotypes

Tau is a microtubule-associated protein whose C-terminal domain consisting of four repeat regions R1, R2, R3 and R4 binds to microtubules to stabilize them. In several neurodegenerative diseases, tau detaches from microtubules to form insoluble aggregates leading to tauopathy. Microtubules are made up of αβ tubulin subunits. Seven α-tubulin and nine β-tubulin isotypes have been reported to be present in humans till date. These tubulin isotypes show residue composition variations mainly at C-terminal region and bind to motor proteins and anti-mitotic drugs differently. These tubulin isotypes show tissue specific expression as their relative proportion varies significantly in different type of cells. It is also known that tau binds differently to different cell lines and can either promote or demote microtubule polymerization. However, the relative binding affinity of tau to the different β-tubulin isotypes present in different cell lines is completely unknown. Here, we study relative binding affinity of Tau repeat region R2 to neuronal specific tubulin isotypes βI, βIIb, and βIII using molecular modelling approach. The order of binding energy of tau with tubulin is βIII > βIIb > βI. Our strategy can be potentially adapted to understand differential binding affinity of tau towards β-tubulin isotypes present in other cell lines.