Tubulin Protein Research Articles

Recent progress of bacterial FtsZ inhibitors with a focus on peptides.

In recent years, the rise of antibiotic resistance has become a primary health problem. With the emergence of bacterial resistance, the need to explore and develop novel antibacterial drugs has become increasingly urgent. Filamentous temperature-sensitive mutant Z (FtsZ), a crucial cell division protein of bacteria, has become a vital antibacterial target. FtsZ is a filamentous GTPase; it is highly conserved in bacteria and shares less than 20% sequence identity with the eukaryotic cytoskeleton protein tubulin, indicating that FtsZ-targeting antibacterial agents may have a low cytotoxicity toward eukaryotes. FtsZ can form a dynamic Z-ring in the center of the cell resulting in cell division. Furthermore, disturbance in the assembly of FtsZ may affect cellular dynamics and bacterial cell survival, making it a fascinating target for drug development. This review focuses on the recent discovery of FtsZ inhibitors, including peptides, natural products, and other synthetic small molecules, as well as their mechanism of action, which could facilitate the discovery of novel FtsZ-targeting clinical drugs in the future.

Quantitative Immunoblotting Analyses Reveal that the Abundance of Actin, Tubulin, Synaptophysin and EEA1 Proteins is Altered in the Brains of Aged Mice

Optimal synaptic activity is essential for cognitive function, including memory and learning. Evidence indicates that cognitive decline in elderly individuals is associated with altered synaptic function. However, the impact of aging on the expression of neurotransmitter receptors and accessory proteins in brain synapses remains unclear. To fill this knowledge gap, we investigated the effect of aging on the mouse brain by utilizing a subcellular brain tissue fractionation procedure to measure protein abundance using quantitative Western Blotting. Comparing 7-month- (control) and 22-month- (aged) old mouse tissue, no significant differences were identified in the levels of AMPA receptor subunits between the experimental groups. The abundance of GluN2B NMDA receptor subunits decreased in aged mice, whereas the levels of GluN2A did not change. The analysis of cytoskeletal proteins showed an altered level of actin and tubulin in aged mice while PSD-95 protein did not change. Vesicle protein analysis revealed that synaptophysin abundance is decreased in older brains whereas EEA1 was significantly increased. Thus, our results suggest that physiological aging profoundly impacts the abundance of molecules associated with neurotransmitter release and vesicle cycling, proteins implicated in cognitive function.

Antiproliferative and antiinflammatory coxib-combretastatin hybrids suppress cell cycle progression and induce apoptosis of MCF7 breast cancer cells.

In our study, some newly synthesized aryl-substituted pyrazole derivatives mimicking cis-diphenylethylene scaffold of two apoptotic inducing agents celecoxib and combretastatin A-4 were found to have strong antiproliferative as well as antiinflammatory activities. Among these coxib-combretastatin hybrids, two lead compounds 8 and 6c simultaneously inhibited prostaglandin E2 (PGE2) production in LPS-activated murine macrophage RAW 264.7 cells and suppressed cell cycle progression of MCF7 cells at G2/M or G0/G1 phases, but only compound 8 induced apoptosis via caspase-3 activation. Both the lead compounds showed good docking energies with both protein targets COX-2 and tubulin in the molecule interaction modeling. The cis-diphenylethylene scaffold of celecoxib or combretastatin A-4 as well as functional groups such as the ethyl ester group and the sulfonamide could be considered as potential key features for the dual activity of studied compounds meanwhile the trimethoxybenzene remained the crucial characterization of the newly derived compounds of combretastatins.

Tubulin Proteins in Cancer Resistance: A Review.

Cancer cells are altered with cell cycle genes or they are mutated, leading to a high rate of proliferation compared to normal cells. Alteration in these genes leads to mitosis dysregulation and becomes the basis of tumor progression and resistance to many drugs. The drugs which act on the cell cycle fail to arrest the process, making cancer cell non-responsive to apoptosis or cell death. Vinca alkaloids and taxanes fall in this category and are referred to as antimitotic agents. Microtubule proteins play an important role in mitosis during cell division as a target site for vinca alkaloids and taxanes. These proteins are dynamic in nature and are composed of α-β-tubulin heterodimers. β-tubulin specially βΙΙΙ isotype is generally altered in expression within cancerous cells. Initially, these drugs were very effective in the treatment of cancer but failed to show their desired action after initial chemotherapy. The present review highlights some of the important targets and their mechanism of resistance offered by cancer cells with new promising drugs from natural sources that can lead to the development of a new approach to chemotherapy.

Structure Based Drug Design and Molecular Docking Studies of Anticancer Molecules Paclitaxel, Etoposide and Topotecan using Novel Ligands.

Tubulin is the biochemical target for several clinically used anticancer drugs as it helps in the formation of mitotic spindle during mitosis stage of cell division. Many of the anti-cancer drugs are known to interact with tubulin and microtubules including some plant alkaloids, such as paclitaxel, etoposide and topotecan. In silico drug design of these molecules were performed prior to testing these drugs in vitro. In silico drug design of these anti-cancer drugs becomes a challenge due to the complex structure of target protein. This challenge was overcome by predicting the structure of the target protein (tubulin) by homology modeling. In this study, computer aided drug designing approach was applied to predict the suitable docking site in target protein and the interaction of tubulin protein with paclitaxel, etoposide and topotecan was explored by molecular docking using Schrödinger software. Docking score and glide energy were determined with ligands to validate their anticancer properties. The results indicate that etoposide is the best drug for tubulin with a docking score of - 4.916 and glide energy of -46.470 kcal/mol compared to paclitaxel and topotecan. The testing of these drugs in silico provides an alternate to in vitro testing of these molecules on cancer cell lines which is a time and cost intensive process. The in silico study of parameters, such as docking score and glide energy, will help pharmacists in developing new molecules as targets for cancers in a time and cost-effective manner.

Recent Developments of Target Based Coumarin Derivatives as Potential Anticancer Agents.

Cancer is the second life-threatening disease worldwide, and it resulted in around 9.6 million deaths globally in 2018. The multidrug-resistant cancers and non-selectivity create an urge for the development of novel anticancer drugs with a diverse mechanism of action. Coumarin is one of the most widely used scaffolds for the development of highly effective anticancer agents. It has versatile anticancer profiles with diverse mechanisms to inhibit tumor progression. The facile synthetic strategies are also favoured for target-based coumarin derivatives. Structural Activity Relationship (SAR) studies determine anticancer potentials with minimal side effects through the substitution pattern of coumarin. A number of coumarin derivatives have been developed against the Galectin-1 (Gal-1), Carbonic anhydrases (CAs), Tubulin protein, and other essential targets for the treatment in cancer therapy. Therefore, in this review, we have mainly focused on different target based coumarin derivatives, and synthetic strategies.

Apoptosis Activation in Thyroid Cancer Cells by Jatrorrhizine-Platinum(II) Complex via Downregulation of PI3K/AKT/Mammalian Target of Rapamycin (mTOR) Pathway.

BackgroundThyroid cancer, which is the most common endocrine cancer, has shown a drastic increase in incidence globally over the past decade. The present study investigated the thyroid cancer-inhibitory potential of jatrorrhizine-platinum(II) complex (JR-P(II) in vitro and in vivo.Material/MethodsThe JR-P(II)-mediated cytotoxicity in thyroid carcinoma cells was determined by using MTT assay. Assessment of acetylated histones, tubulin, and DNA repair proteins was made by Western blot assays. Flow cytometry was used for apoptosis and ROS accumulation measurement.ResultsThe JR-P(II) suppressed proliferative capacity of SW1736, BHP7-13, and 8305C cells. JR-P(II) treatment markedly promoted expression of acetylated histone H3, H4, and tubulin in a dose-dependent manner. Treatment with JR-P(II) significantly elevated the proportion of cells in sub-G1 and promoted cleaved caspase-3 and -9. In JR-P(II)-treated cells, DCFH-DA fluorescence was much higher relative to control cells. The JR-P(II) treatment consistently suppressed expression of pS6, p-ERK1/2, p-4E-BP1, and p-AKT, and increased p-H2AX expression and suppressed KU70 and KU80 protein levels. The level of RAD51 was repressed in JR-P(II)-treated cells. JR-P(II) administration in mice caused no significant change in body weight, and it inhibited SW1736 tumor growth in mice.ConclusionsThe JR-P(II) induced cytotoxicity in thyroid cancer cells by inhibiting the mechanism responsible for repair of double-stranded DNA. The in vivo data also revealed that JR-P(II) effectively inhibits thyroid tumor growth by inducing DNA damage. Thus, our results suggest that further evaluation of JR-P(II) as a therapeutic candidate for thyroid cancer is warranted.

Soft mechano-chemistry of molecular hubs in mitotic spindle: biomechanics and mechanical proofreading at microtubule ends

A microtubule (MT) is a long stiff tube-shaped filament formed by a hierarchical organization of a large number of tubulin protein molecules. These filaments constitute a major structural component of the scaffold of a multi-component macromolecular machine called mitotic spindle. The plus ends of the MTs are tethered to some specific binding partners by molecular tethers while those of some others are crosslinked by crosslinking molecules. Because of the non-covalent binding involved in the tethering and crosslinking, the attachments formed are intrinsically ‘soft’. These attachments are transient because these can get ruptured spontaneously by thermal fluctuations. By implementing in silico the standard protocols of in vitro molecular force spectroscopy, we compute the lifetimes of simple theoretical models of these attachments. The mean lifetime is essentially a mean first-passage time. The stability of cross-linked antiparallel MTs is shown to decrease monotonically with increasing tension, a characteristic of all ‘slip-bonds’. This is in sharp contrast to the nonmonotonic variation of the mean lifetime with tension, a mechanical fingerprint of ‘catch-bonds’, displayed by the MTs tethered to two distinct binding partners. We mention plausible functional implications of these observations in the context of mechanical proofreading.

Fractal, Scale Free Electromagnetic Resonance of a Single Brain Extracted Microtubule Nanowire, a Single Tubulin Protein and a Single Neuron

Biomaterials are primarily insulators. For nearly a century, electromagnetic resonance and antenna–receiver properties have been measured and extensively theoretically modeled. The dielectric constituents of biomaterials—if arranged in distinct symmetries, then each vibrational symmetry—would lead to a distinct resonance frequency. While the literature is rich with data on the dielectric resonance of proteins, scale-free relationships of vibrational modes are scarce. Here, we report a self-similar triplet of triplet resonance frequency pattern for the four-4 nm-wide tubulin protein, for the 25-nm-wide microtubule nanowire and 1-μm-wide axon initial segment of a neuron. Thus, preserving the symmetry of vibrations was a fundamental integration feature of the three materials. There was no self-similarity in the physical appearance: the size varied by 106 orders, yet, when they vibrated, the ratios of the frequencies changed in such a way that each of the three resonance frequency bands held three more bands inside (triplet of triplet). This suggests that instead of symmetry, self-similarity lies in the principles of symmetry-breaking. This is why three elements, a protein, it’s complex and neuron resonated in 106 orders of different time domains, yet their vibrational frequencies grouped similarly. Our work supports already-existing hypotheses for the scale-free information integration in the brain from molecular scale to the cognition.

Protein Complex NDC80: Properties, Functions, and Possible Role in Pathophysiology of Cell Division.

Mitotic division maintains genetic identity of any multicellular organism throughout an entire lifetime. Each time a parent cell divides, chromosomes are equally distributed between the daughter cells due to the action of mitotic spindle. Mitotic spindle is formed by the microtubules that represent dynamic polymers of tubulin protein. Spindle microtubules are attached end-on to kinetochores - large multi-protein complexes on chromosomes. This review focuses on the four-subunit NDC80 complex, one of the most important kinetochore elements that plays a major role in the attachment of assembling/disassembling microtubule ends to the chromosomes. Here, we summarize published data on the structure, properties, and regulation of the NDC80 complex and discuss possible relationship between changes in the expression of genes coding for the NDC80 complex components, mitotic disorders, and oncogenesis with special emphasis on the diagnostic and therapeutic potential of NDC80.

Towards further Understanding the Structural Requirements of Combretastatin- like Chalcones as Inhibitors of Microtubule Polymerization.

Microtubules are dynamic filamentous cytoskeletal structures which play several key roles in cell proliferation and trafficking. They are supposed to contribute in the development of important therapeutic targeting tumor cells. Chalcones are important group of natural compounds abundantly found in fruits & vegetables that are known to possess anticancer activity. We have used QSAR and docking studies to understand the structural requirement of chalcones for understanding the mechanism of microtubule polymerization inhibition. Three dimensional (3D) QSAR (CoMFA and CoMSIA), pharmacophore mapping and molecular docking studies were performed for the generation of structure activity relationship of combretastatin-like chalcones through statistical models and contour maps. Structure activity relationship revealed that substitution of electrostatic, steric and donor groups may enhance the biological activity of compounds as inhibitors of microtubule polymerization. From the docking study, it was clear that compounds bind at the active site of tubulin protein. The given strategies of modelling could be an encouraging way for designing more potent compounds as well as for the elucidation of protein-ligand interaction.

The Molecular Chaperone CCT/TRiC: An Essential Component of Proteostasis and a Potential Modulator of Protein Aggregation.

Chaperonin containing tailless complex polypeptide 1 (CCT) or tailless complex polypeptide 1 ring complex (TRiC) is an essential eukaryotic molecular chaperone. It is a multi-subunit oligomer of two rings of eight individual protein subunits. When assembled, each of the eight CCT subunits occupies a specific position within each chaperonin ring. Thus a geometrically defined binding interface is formed from the divergent sequences within the CCT subunit substrate binding domains. CCT is required for the folding of the abundant cytoskeletal proteins actin and tubulin, which in turn form assemblies of microfilaments and microtubules. CCT is also involved in the folding of some additional protein substrates and some CCT subunits have been shown to have functions when monomeric. Since observations were made in worms over a decade ago using an RNAi screen, which connected CCT subunits to the aggregation of polyglutamine tracts, a role for CCT as a potential modulator of protein aggregation has started to emerge. Here there will be a focus on how mechanistically CCT may be able to achieve this and if this potential function of CCT provides any insights and directions for developing future treatments for protein aggregation driven neurodegenerative diseases generally, many of which are associated with aging.

Anticancer Potential of N‐Sulfonyl Noscapinoids: Synthesis and Evaluation

AbstractNoscapine is an alkaloid with a basic phthalideisoquinoline moiety and is well recognized as an antitussive drug. It is obtained from opium poppy Papaver somniferum and demonstrates a rather safe toxicity profile in vitro. Recent studies found noscapine and its analogues to have anticancer activity against mammalian cancer cell lines by modulating microtubule assembly. In the present study, we report the synthesis and evaluation of cytotoxicity of a series of N‐sulfonyl noscapinoids 6 a–n obtained by reaction of nornoscapine with various aryl/heteroaryl sulfonyl chlorides. To assess the antiproliferative activity of the novel derivatives 6 a–n, SRB assay was performed in four different human cancer cell lines, MIAPaCa‐2 (pancreatic), HeLa (cervical), SK−N−SH (neuroblastoma) and DU145 (prostate cancer). The study identified four compounds 6 e–g and 6 j ability to arrest cell‐cycle at the G2/M phase and cytotoxic potential against the human pancreatic cancer cell line MIAPaCa‐2. We observed that these compounds 6 e–g and 6 j induce microtubule depolymerisation and cause cell cycle arrest at the G2/M phase thereby resulting in caspase‐3 and PARP activation leading to cell death. In silico molecular docking studies of these compounds showed non‐covalent interactions like Van der Waals and hydrogen‐bonding with tubulin protein and with good binding energy.

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.

Posterior Neocortex-Specific Regulation of Neuronal Migration by CEP85L Identifies Maternal Centriole-Dependent Activation of CDK5

Genes mutated in human neuronal migration disorders encode tubulin proteins and a variety of tubulin-binding and -regulating proteins, but it is very poorly understood how these proteins function together to coordinate migration. Additionally, the way in which regional differences in neocortical migration are controlled is completely unknown. Here we describe a new syndrome with remarkably region-specific effects on neuronal migration in the posterior cortex, reflecting de novo variants in CEP85L. We show that CEP85L is required cell autonomously invivo and invitro for migration, that it localizes to the maternal centriole, and that it forms a complex with many other proteins required for migration, including CDK5, LIS1, NDE1, KIF2A, and DYNC1H1. Loss of CEP85L disrupts CDK5 localization and activation, leading to centrosome disorganization and disrupted microtubule cytoskeleton organization. Together, our findings suggest that CEP85L highlights a complex that controls CDK5 activity to promote neuronal migration.

Unraveling the molecular mechanism of BNC105, a phase II clinical trial vascular disrupting agent, provides insights into drug design

Microtubules are made up of tubulin protein and play a very important part in numerous cellular events of eukaryotic cells, which is why they are seen as attractive targets for tumor chemotherapy. BNC105, a known vascular targeting agent, has entered in phase II clinical trials. It has previously been confirmed that BNC105 is an effective microtubule targeting agent for various cancers. BNC105 exhibits selectivity for tumor cells, elicits vascular disrupting effects, and inhibits tumor growth. However, the molecular mechanism of BNC105 is still elusive. Herein, the crystal structure of BNC105 in complex with tubulin protein is revealed, demonstrating the its interaction with the colchicine binding site. In order to thoroughly evaluate its molecular mechanism from a structural-activity-relationship standpoint, the binding mode of tubulin to BNC-105 is compared with colchicine, CA-4 and other BNC-105 derivatives. Our study not only confirms the detailed interactions of the BNC105-tubulin complex, but also offer substantial structural foundation for the design and development of novel benzo[b]furan derivatives as microtubule targeting agents.

Detection of Tubulin and TAU Proteins Aggregations using Solid-State Nanopore and Atomic Force Microscopy (AFM)

Aggregations of tau and tubulin proteins are one of the leading causes of many neurodegenerative diseases, such as Pick's, Alzheimer's, and Parkinson's. In this work, protein aggregations are analyzed in salt solution using a solid-state nanopore sensing system. A varieties of fabrication techniques, such as photolithography, chemical vapor deposition, focused ion beam milling and ion beam sculpting are used to fabricate 8 - 30 nm nanopores in free-standing silicon nitride membrane on silicon. The nanopore is placed in between two polydimethylsiloxane chambers with microfluidic channels filled with 1 M KCl solution, and a pair of silver chloride electrodes are embedded in the channels. After a stable ionic current is established by applying bias voltage across the electrodes, charged protein molecules are added to the top chamber (ground), and driven to the bottom chamber by the applied electric field. The passing protein molecules partially block the ion flow in the nanopore and increase the pore resistivity, as a result the ionic current drops. The amplitude of the current drops is proportional to the size of the protein molecules. In this work, the aggregations of tau and tubulin proteins are detected at bias voltages ranges from 60 - 210 mV and pH ranges from 7.4 - 12.0. Monomer proteins were measured with 8 - 10 nm nanopores, aggregated proteins were measured using 15 - 30 nm nanopores. Finally, the amplitude of current drops for monomer and aggregated proteins are compared. Our results show that the tau and tubulin proteins are aggregated to dimer, trimer and multimers. The nanopore results are verified by AFM scanning of dried protein samples on mica surface. The AFM results are found to be consistent with the nanopore measurements.

Interplay between Convective and Viscoelastic Forces Controls the Morphology of In Vitro Paclitaxel-Stabilized Microtubules

Microtubules (MTs) are self-assembling, high-aspect-ratio tubular nanostructures formed from the polymerization of tubulin protein. MTs are capable of globally assembling into optically birefringent morphologies, but there is disagreement on the mechanisms driving this behavior. We investigated the temporal evolution of paclitaxel (PTX)-stabilized MT solutions under a range of in vitro conditions. Significant morphological differences were observed in the polymerized PTX-MT solutions as a consequence of varying the orientation of the reaction vessel (vertical vs. horizontal), the type of heating source (hot plate vs. incubator), the incubation time, and the concentration of PTX (high vs. low). The most robust birefringent patterns were found only in vertically oriented cuvettes that were heated asymmetrically on a hot plate, suggesting dependence upon a convective flow, which we confirmed with a combination of optical and thermal imaging. Higher concentrations of PTX led to denser PTX-MT domain formation and brighter birefringence, due to more complete polymerization. Combining our experimental observations, we conclude that birefringent patterns arise principally through a combination of convective and viscoelastic forces, and we identify the sequence of dynamical stages through which they evolve.

A lower affinity to cytosolic proteins reveals VDAC3 isoform-specific role in mitochondrial biology.

Voltage-dependent anion channel (VDAC) is the major pathway for the transport of ions and metabolites across the mitochondrial outer membrane. Among the three known mammalian VDAC isoforms, VDAC3 is the least characterized, but unique functional roles have been proposed in cellular and animal models. Yet, a high-sequence similarity between VDAC1 and VDAC3 is indicative of a similar pore-forming structure. Here, we conclusively show that VDAC3 forms stable, highly conductive voltage-gated channels that, much like VDAC1, are weakly anion selective and facilitate metabolite exchange, but exhibit unique properties when interacting with the cytosolic proteins α-synuclein and tubulin. These two proteins are known to be potent regulators of VDAC1 and induce similar characteristic blockages (on the millisecond time scale) of VDAC3, but with 10- to 100-fold reduced on-rates and altered α-synuclein blocking times, indicative of an isoform-specific function. Through cysteine scanning mutagenesis, we found that VDAC3's cysteine residues regulate its interaction with α-synuclein, demonstrating VDAC3-unique functional properties and further highlighting a general molecular mechanism for VDAC isoform-specific regulation of mitochondrial bioenergetics.

Structures of \u03b2-glycosidase LXYL-P1-2 reveals the product binding state of GH3 family and a specific pocket for Taxol recognition

LXYL-P1-2 is one of the few xylosidases that efficiently catalyze the reaction from 7-β-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT), and is a potential enzyme used in Taxol industrial production. Here we report the crystal structure of LXYL-P1-2 and its XDT binding complex. These structures reveal an enzyme/product complex with the sugar conformation different from the enzyme/substrate complex reported previously in GH3 enzymes, even in the whole glycohydrolases family. In addition, the DT binding pocket is identified as the structural basis for the substrate specificity. Further structure analysis reveals common features in LXYL-P1-2 and Taxol binding protein tubulin, which might provide useful information for designing new Taxol carrier proteins for drug delivery.