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Science (New York, N.Y.) Jan 2020Tubulins play crucial roles in cell division, intracellular traffic, and cell shape. Tubulin concentration is autoregulated by feedback control of messenger RNA (mRNA)...
Tubulins play crucial roles in cell division, intracellular traffic, and cell shape. Tubulin concentration is autoregulated by feedback control of messenger RNA (mRNA) degradation via an unknown mechanism. We identified tetratricopeptide protein 5 (TTC5) as a tubulin-specific ribosome-associating factor that triggers cotranslational degradation of tubulin mRNAs in response to excess soluble tubulin. Structural analysis revealed that TTC5 binds near the ribosome exit tunnel and engages the amino terminus of nascent tubulins. TTC5 mutants incapable of ribosome or nascent tubulin interaction abolished tubulin autoregulation and showed chromosome segregation defects during mitosis. Our findings show how a subset of mRNAs can be targeted for coordinated degradation by a specificity factor that recognizes the nascent polypeptides they encode.
Topics: Feedback, Physiological; HEK293 Cells; Humans; Mutation; Protein Biosynthesis; RNA Stability; RNA, Messenger; Ribosomes; Transcription Factors; Tubulin
PubMed: 31727855
DOI: 10.1126/science.aaz4352 -
Cell Dec 2022The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we...
The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.
Topics: Humans; Chaperonin Containing TCP-1; Tubulin; Protein Folding; Proteostasis; Adenosine Triphosphate
PubMed: 36493755
DOI: 10.1016/j.cell.2022.11.014 -
Cells Mar 2021Members of the tubulin superfamily are GTPases; the activities of GTPases are necessary for life. The members of the tubulin superfamily are the constituents of the... (Review)
Review
Members of the tubulin superfamily are GTPases; the activities of GTPases are necessary for life. The members of the tubulin superfamily are the constituents of the microtubules and the γ-tubulin meshwork. Mutations in members of the tubulin superfamily are involved in developmental brain disorders, and tubulin activities are the target for various chemotherapies. The intricate functions (game) of tubulins depend on the activities of the GTP-binding domain of α-, β-, and γ-tubulin. This review compares the GTP-binding domains of γ-tubulin, α-tubulin, and β-tubulin and, based on their similarities, recapitulates the known functions and the impact of the γ-tubulin GTP-binding domain in the regulation of the γ-tubulin meshwork and cellular homeostasis.
Topics: Amino Acid Sequence; Animals; Guanosine Triphosphate; Humans; Microtubules; Phosphorylation; Protein Domains; Tubulin
PubMed: 33800665
DOI: 10.3390/cells10040745 -
Neuropsychopharmacology Reports Mar 2022Clozapine is currently the only effective drug for treatment-resistant schizophrenia; nonetheless, its pharmacological mechanism remains unclear, and its administration...
AIM
Clozapine is currently the only effective drug for treatment-resistant schizophrenia; nonetheless, its pharmacological mechanism remains unclear, and its administration is limited because of severe adverse effects. By comparing the binding proteins of clozapine and its derivative olanzapine, which is safer but less effective than clozapine, we attempted to clarify the mechanism of action specific to clozapine.
METHODS
First, using the polyproline rod conjugates attached with clozapine or olanzapine, clozapine-binding proteins in extracts from the cerebra of 7-week-old ICR mice were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins. Second, the effect of clozapine on tubulin polymerization was determined turbidimetrically. Finally, the cellular effects of clozapine were observed in HeLa cells by immunofluorescence microscopy.
RESULTS
Alpha and β tubulins were the most abundant clozapine-binding proteins. We also found that clozapine directly binds with α and β tubulin heterodimers to inhibit their polymerization to form microtubules and disturbs the microtubule network, causing mitotic arrest in HeLa cells.
CONCLUSION
These results suggest that α and β tubulin heterodimers are targeted by the clozapine and the microtubules are involved in the etiology of schizophrenia.
Topics: Animals; Chromatography, Liquid; Clozapine; HeLa Cells; Humans; Mice; Mice, Inbred ICR; Microtubules; Tandem Mass Spectrometry; Tubulin
PubMed: 34964309
DOI: 10.1002/npr2.12221 -
Biochimica Et Biophysica Acta Jul 2009The association of tubulin with the plasma membrane comprises multiple levels of penetration into the bilayer: from integral membrane protein, to attachment via... (Review)
Review
The association of tubulin with the plasma membrane comprises multiple levels of penetration into the bilayer: from integral membrane protein, to attachment via palmitoylation, to surface binding, and to microtubules attached by linker proteins to proteins in the membrane. Here we discuss the soundness and weaknesses of the chemical and biochemical evidence marshaled to support these associations, as well as the mechanisms by which tubulin or microtubules may regulate functions at the plasma membrane.
Topics: Cell Membrane; Humans; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Tubulin
PubMed: 19328773
DOI: 10.1016/j.bbamem.2009.03.013 -
Methods in Cell Biology 2010New analytical methods are needed for the successful outcome of experiments aimed at characterizing mechanisms of microtubule dynamics and at understanding the effects... (Review)
Review
New analytical methods are needed for the successful outcome of experiments aimed at characterizing mechanisms of microtubule dynamics and at understanding the effects of drugs on microtubules. The identification of tubulin isotypes and of regions of the microtubule involved in drug interactions has been advanced by proteomic methodologies. The diversity of tubulin sequences and posttranslational modifications (PTMs) can generate a complex mixture of heterodimers with unique molecular dynamics driving specific functions. Mass spectrometry (MS)-based approaches have been developed, and in combination with chromatographic and/or electrophoretic separation of tubulin polypeptides or peptides, they have contributed to our understanding of tubulin proteomics. We present protocols that we have used for the analysis of tubulin isotypes and PTMs present in tubulin isolated from cells in culture or tissues and for the identification of tubulin regions altered by microtubule-stabilizing agents. Tubulin proteomics complements structural and computer modeling information for a high-resolution view of microtubule dynamics and its alteration by drugs. These methodologies will help in providing insights into tubulin isotype-specific functions and in the design of drugs targeting either all tubulin heterodimers indiscriminately or only those containing specific isotypes.
Topics: Animals; Cells; Humans; Microtubule Proteins; Pharmaceutical Preparations; Protein Binding; Protein Isoforms; Protein Processing, Post-Translational; Proteomics; Tissue Extracts; Tubulin
PubMed: 20466132
DOI: 10.1016/S0091-679X(10)95007-3 -
BioEssays : News and Reviews in... Jul 2007The eukaryotic cytoskeleton appears to have evolved from ancestral precursors related to prokaryotic FtsZ and MreB. FtsZ and MreB show 40-50% sequence identity across... (Review)
Review
The eukaryotic cytoskeleton appears to have evolved from ancestral precursors related to prokaryotic FtsZ and MreB. FtsZ and MreB show 40-50% sequence identity across different bacterial and archaeal species. Here I suggest that this represents the limit of divergence that is consistent with maintaining their functions for cytokinesis and cell shape. Previous analyses have noted that tubulin and actin are highly conserved across eukaryotic species, but so divergent from their prokaryotic relatives as to be hardly recognizable from sequence comparisons. One suggestion for this extreme divergence of tubulin and actin is that it occurred as they evolved very different functions from FtsZ and MreB. I will present new arguments favoring this suggestion, and speculate on pathways. Moreover, the extreme conservation of tubulin and actin across eukaryotic species is not due to an intrinsic lack of variability, but is attributed to their acquisition of elaborate mechanisms for assembly dynamics and their interactions with multiple motor and binding proteins. A new structure-based sequence alignment identifies amino acids that are conserved from FtsZ to tubulins. The highly conserved amino acids are not those forming the subunit core or protofilament interface, but those involved in binding and hydrolysis of GTP.
Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Cytoskeletal Proteins; Cytoskeleton; Evolution, Molecular; Humans; Molecular Sequence Data; Sequence Homology, Amino Acid; Tubulin
PubMed: 17563102
DOI: 10.1002/bies.20601 -
Drug Discovery Today Mar 2022Tubulin is an important cancer drug target. Compounds that bind at the colchicine site in tubulin have attracted significant interest as they are generally less affected... (Review)
Review
Tubulin is an important cancer drug target. Compounds that bind at the colchicine site in tubulin have attracted significant interest as they are generally less affected by multidrug resistance than other potential drugs. Modeling is useful in understanding the interactions between tubulin and colchicine binding site inhibitors (CBSIs), but because the colchicine binding site contains two flexible loops whose conformations are highly ligand-dependent, modeling has its limitations. X-ray crystallography provides experimental pictures of tubulin-ligand interactions at this challenging colchicine site. Since 2004, when the first X-ray structure of tubulin in complex with N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine) was published, many X-ray crystal structures have been reported for tubulin complexes involving the colchicine binding site. In this review, we summarize the crystal structures of tubulin in complexes with various CBSIs, aiming to facilitate the discovery of new generations of tubulin inhibitors.
Topics: Binding Sites; Colchicine; Crystallography, X-Ray; Ligands; Tubulin; Tubulin Modulators
PubMed: 34890803
DOI: 10.1016/j.drudis.2021.12.001 -
Angewandte Chemie (International Ed. in... Jun 2021Tubulin plays essential roles in vital cellular activities and is the target of a wide range of proteins and ligands. Here, using a combined computational and...
Tubulin plays essential roles in vital cellular activities and is the target of a wide range of proteins and ligands. Here, using a combined computational and crystallographic fragment screening approach, we addressed the question of how many binding sites exist in tubulin. We identified 27 distinct sites, of which 11 have not been described previously, and analyzed their relationship to known tubulin-protein and tubulin-ligand interactions. We further observed an intricate pocket communication network and identified 56 chemically diverse fragments that bound to 10 distinct tubulin sites. Our results offer a unique structural basis for the development of novel small molecules for use as tubulin modulators in basic research applications or as drugs. Furthermore, our method lays down a framework that may help to discover new pockets in other pharmaceutically important targets and characterize them in terms of chemical tractability and allosteric modulation.
Topics: Allosteric Regulation; Binding Sites; Crystallography, X-Ray; Ligands; Molecular Dynamics Simulation; Protein Binding; Tubulin; Tubulin Modulators
PubMed: 33951246
DOI: 10.1002/anie.202100273 -
Proceedings of the National Academy of... Aug 2022Cilia are microtubule-based organelles that power cell motility and regulate sensation and signaling, and abnormal ciliary structure and function cause various...
Cilia are microtubule-based organelles that power cell motility and regulate sensation and signaling, and abnormal ciliary structure and function cause various ciliopathies. Cilium formation and maintenance requires intraflagellar transport (IFT), during which the kinesin-2 family motor proteins ferry IFT particles carrying axonemal precursors such as tubulins into cilia. Tubulin dimers are loaded to IFT machinery through an interaction between tubulin and the IFT-74/81 module; however, little is known of how tubulins are unloaded when arriving at the ciliary tip. Here, we show that the ciliary kinase DYF-5/MAK phosphorylates multiple sites within the tubulin-binding module of IFT-74, reducing the tubulin-binding affinity of IFT-74/81 approximately sixfold. Ablation or constitutive activation of IFT-74 phosphorylation abnormally elongates or shortens sensory cilia in neurons. We propose that DYF-5/MAK-dependent phosphorylation plays a fundamental role in ciliogenesis by regulating tubulin unloading.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cilia; Mitogen-Activated Protein Kinases; Phosphorylation; Tubulin
PubMed: 35969738
DOI: 10.1073/pnas.2207134119