scholarly journals Effects of α-tubulin acetylation on microtubule structure and stability

2019 ◽  
Author(s):  
Lisa Eshun-Wilson ◽  
Rui Zhang ◽  
Didier Portran ◽  
Maxence Nachury ◽  
Dan Toso ◽  
...  
Keyword(s):  
Conformational Sampling ◽  
Post Translational Modification ◽  
Conformational Ensembles ◽  
Microtubule Stabilization ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Cryo Electron Microscopy ◽  
Conformational Landscape ◽  
Dynamics Simulations ◽  
And Function

ABSTRACTAcetylation of K40 in α-tubulin is the sole post-translational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as the structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.

scholarly journals Effects of α-tubulin acetylation on microtubule structure and stability

2019 ◽  
Vol 116 (21) ◽  
pp. 10366-10371 ◽  
Author(s):  
Lisa Eshun-Wilson ◽  
Rui Zhang ◽  
Didier Portran ◽  
Maxence V. Nachury ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Conformational Sampling ◽  
Conformational Ensembles ◽  
Microtubule Stabilization ◽  
Flexible Loop ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Cryo Electron Microscopy ◽  
Conformational Landscape ◽  
Dynamics Simulations ◽  
And Function

Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.

scholarly journals The ribosome and its role in protein folding: looking through a magnifying glass

2017 ◽  
Vol 73 (6) ◽  
pp. 509-521 ◽  
Author(s):  
Abid Javed ◽  
John Christodoulou ◽  
Lisa D. Cabrita ◽  
Elena V. Orlova
Keyword(s):  
Protein Folding ◽  
Structural Biology ◽  
Polypeptide Chain ◽  
Structure And Function ◽  
Cellular Activity ◽  
Cryo Electron Microscopy ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Dynamics Simulations ◽  
And Function

Protein folding, a process that underpins cellular activity, begins co-translationally on the ribosome. During translation, a newly synthesized polypeptide chain enters the ribosomal exit tunnel and actively interacts with the ribosome elements – the r-proteins and rRNA that line the tunnel – prior to emerging into the cellular milieu. While understanding of the structure and function of the ribosome has advanced significantly, little is known about the process of folding of the emerging nascent chain (NC). Advances in cryo-electron microscopy are enabling visualization of NCs within the exit tunnel, allowing early glimpses of the interplay between the NC and the ribosome. Once it has emerged from the exit tunnel into the cytosol, the NC (still attached to its parent ribosome) can acquire a range of conformations, which can be characterized by NMR spectroscopy. Using experimental restraints within molecular-dynamics simulations, the ensemble of NC structures can be described. In order to delineate the process of co-translational protein folding, a hybrid structural biology approach is foreseeable, potentially offering a complete atomic description of protein folding as it occurs on the ribosome.

scholarly journals SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Mohsen Sarikhani ◽  
Sneha Mishra ◽  
Sangeeta Maity ◽  
Chaithanya Kotyada ◽  
Donald Wolfgeher ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Serine Phosphorylation ◽  
Glycogen Synthase Kinase 3 ◽  
Lysine Acetylation ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Dynamics Simulations ◽  
And Function ◽  
Reduced Activity ◽  
Deficient Mice

Glycogen synthase kinase 3 (GSK3) is a critical regulator of diverse cellular functions involved in the maintenance of structure and function. Enzymatic activity of GSK3 is inhibited by N-terminal serine phosphorylation. However, alternate post-translational mechanism(s) responsible for GSK3 inactivation are not characterized. Here, we report that GSK3α and GSK3β are acetylated at Lys246 and Lys183, respectively. Molecular modeling and/or molecular dynamics simulations indicate that acetylation of GSK3 isoforms would hinder both the adenosine binding and prevent stable interactions of the negatively charged phosphates. We found that SIRT2 deacetylates GSK3β, and thus enhances its binding to ATP. Interestingly, the reduced activity of GSK3β is associated with lysine acetylation, but not with phosphorylation at Ser9 in hearts of SIRT2-deficient mice. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2 in cardiomyocytes. Overall, our study identified lysine acetylation as a novel post-translational modification regulating GSK3 activity.

A Robotics Approach to Enhance Conformational Sampling of Proteins

2012 ◽  
Author(s):  
Juan Cortés ◽  
Ibrahim Al-Bluwi
Keyword(s):  
Monte Carlo ◽  
Computational Methods ◽  
Conformational Space ◽  
Conformational Sampling ◽  
Biological Macromolecules ◽  
Dynamics And Function ◽  
Living Organisms ◽  
Dynamics Simulations ◽  
And Function ◽  
The Relationship

Proteins are biological macromolecules that play essential roles in living organisms. Furthermore, the study of proteins and their function is of interest in other fields in addition to biology, such as pharmacology and biotechnology. Understanding the relationship between protein structure, dynamics and function is indispensable for advances in all these areas. This requires a combination of experimental and computational methods, whose development is the object of very active interdisciplinary research. In such a context, this paper presents a technique to enhance conformational sampling of proteins carried out with computational methods such as molecular dynamics simulations or Monte Carlo methods. Our approach is based on a mechanistic representation of proteins that enables the application of efficient methods originating from robotics. The paper explains the generalities of the approach, and gives details on its application to devise Monte Carlo move classes. Results show the good performance of the method for sampling the conformational space of different types of proteins.

Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau

1992 ◽  
Vol 103 (4) ◽  
pp. 953-964 ◽  
Author(s):  
R. Takemura ◽  
S. Okabe ◽  
T. Umeyama ◽  
Y. Kanai ◽  
N.J. Cowan ◽  
...  
Keyword(s):  
Post Translational Modification ◽  
3T3 Cells ◽  
Microtubule Organization ◽  
Alpha Tubulin ◽  
Transfected Cells ◽  
Microtubule Associated Proteins ◽  
Cos Cells ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Associated Proteins

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.

scholarly journals Posttranslational Modifications of Tubulin and the Polarized Transport of Kinesin-1 in Neurons

2010 ◽  
Vol 21 (4) ◽  
pp. 572-583 ◽  
Author(s):  
Jennetta W. Hammond ◽  
Chun-Fang Huang ◽  
Stefanie Kaech ◽  
Catherine Jacobson ◽  
Gary Banker ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Glycogen Synthase ◽  
Morphological Differentiation ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Polarized Transport ◽  
And Function ◽  
Selective Accumulation

Polarized transport by microtubule-based motors is critical for neuronal development and function. Selective translocation of the Kinesin-1 motor domain is the earliest known marker of axonal identity, occurring before morphological differentiation. Thus, Kinesin-1–mediated transport may contribute to axonal specification. We tested whether posttranslational modifications of tubulin influence the ability of Kinesin-1 motors to distinguish microtubule tracks during neuronal development. We detected no difference in microtubule stability between axons and minor neurites in polarized stage 3 hippocampal neurons. In contrast, microtubule modifications were enriched in a subset of neurites in unpolarized stage 2 cells and the developing axon in polarized stage 3 cells. This enrichment correlated with the selective accumulation of constitutively active Kinesin-1 motors. Increasing tubulin acetylation, without altering the levels of other tubulin modifications, did not alter the selectivity of Kinesin-1 accumulation in polarized cells. However, globally enhancing tubulin acetylation, detyrosination, and polyglutamylation by Taxol treatment or inhibition of glycogen synthase kinase 3β decreased the selectivity of Kinesin-1 translocation and led to the formation of multiple axons. Although microtubule acetylation enhances the motility of Kinesin-1, the preferential translocation of Kinesin-1 on axonal microtubules in polarized neuronal cells is not determined by acetylation alone but is probably specified by a combination of tubulin modifications.

scholarly journals New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Bitam ◽  
Ahmad Elbahnsi ◽  
Geordie Creste ◽  
Iwona Pranke ◽  
Benoit Chevalier ◽  
...  
Keyword(s):  
Phosphinic Acid ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transmembrane Conductance Regulator ◽  
Intracellular Loop ◽  
Respiratory Cells ◽  
Cftr Protein ◽  
Dynamics Simulations ◽  
And Function ◽  
Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

scholarly journals Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

2021 ◽  
Vol 11 (9) ◽  
pp. 4048
Author(s):  
Javier A. Linares-Pastén ◽  
Lilja Björk Jonsdottir ◽  
Gudmundur O. Hreggvidsson ◽  
Olafur H. Fridjonsson ◽  
Hildegard Watzlawick ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Ligand Docking ◽  
Glycoside Hydrolase Family ◽  
Ligand Interactions ◽  
Tim Barrel ◽  
Branching Point ◽  
The Difference ◽  
Dynamics Simulations ◽  
And Function ◽  
Hydrolase Family

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

scholarly journals Sumoylation regulates the assembly and activity of the SMN complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Giulietta M. Riboldi ◽  
Irene Faravelli ◽  
Takaaki Kuwajima ◽  
Nicolas Delestrée ◽  
Georgia Dermentzaki ◽  
...  
Keyword(s):  
Survival Rate ◽  
Muscular Atrophy ◽  
Motor Deficits ◽  
Cellular Distribution ◽  
Post Translational Modification ◽  
Smn Complex ◽  
Sensory Motor ◽  
Motor Neuron Loss ◽  
And Function ◽  
Small Nuclear Ribonucleoproteins

AbstractSMN is a ubiquitously expressed protein and is essential for life. SMN deficiency causes the neurodegenerative disease spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. SMN interacts with itself and other proteins to form a complex that functions in the assembly of ribonucleoproteins. SMN is modified by SUMO (Small Ubiquitin-like Modifier), but whether sumoylation is required for the functions of SMN that are relevant to SMA pathogenesis is not known. Here, we show that inactivation of a SUMO-interacting motif (SIM) alters SMN sub-cellular distribution, the integrity of its complex, and its function in small nuclear ribonucleoproteins biogenesis. Expression of a SIM-inactivated mutant of SMN in a mouse model of SMA slightly extends survival rate with limited and transient correction of motor deficits. Remarkably, although SIM-inactivated SMN attenuates motor neuron loss and improves neuromuscular junction synapses, it fails to prevent the loss of sensory-motor synapses. These findings suggest that sumoylation is important for proper assembly and function of the SMN complex and that loss of this post-translational modification impairs the ability of SMN to correct selective deficits in the sensory-motor circuit of SMA mice.

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