BisG10, a K+ channel blocker, affects the calcium release channel from skeletal muscle sarcoplasmic reticulum

AbstractThe action of bisG10, a potent K+ channel inhibitor, was tested on the Ca2+ release from isolated sarcoplasmic reticulum vesicles of rabbit skeletal muscle. Using a rapid filtration technique, we found that the drug inhibited Ca2+-induced Ca2+ release elicited in the presence of extravesicular K+ as counter-ion. This inhibition was not reversed by the addition of valinomycin and still occurred when Cl− was used as co-ion, indicating that not only K+ channels are involved in the inhibiting effect. We found that bisG10 decreased the binding of ryanodine to sarcoplasmic reticulum vesicles, showing that bisG10 is able to block the sarcoplasmic reticulum Ca2+ release channel

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process

Evidence for new C-terminally truncated variants of α- and β-tubulins

New C-terminally truncated α- and β-tubulin variants, both ending with an -EEEG sequence, are identified in vivo: αΔ3-tubulin, which has a specific neuronal distribution pattern (distinct from that of αΔ2-tubulin) and seems to be related to dynamic microtubules, and βΔ4-tubulin, corresponding to β2A/B-tubulin modified by truncation of four C-terminal residues, which is ubiquitously present in cells and tissues. Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease

: Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions which go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase. Here we show that tubulin tyrosine ligase hemizygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density, and both synaptic plasticity and memory deficits. We further report decreased tubulin tyrosine ligase expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harboring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid β peptide toxicity and that expression of tubulin tyrosine ligase, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid β peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid β peptide-induced synaptic damage, and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease

Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin

Proteomic and Functional Studies Reveal Detyrosinated Tubulin as Treatment Target in Sarcomere Mutation-Induced Hypertrophic Cardiomyopathy

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ≈50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, HCMSMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, HCMSMN). Genotype-specific differences have been reported in cardiac function. Moreover, HCMSMN patients have later disease onset and a better prognosis than HCMSMP patients. To define if genotype-specific derailments at the protein level may explain the heterogeneity in disease development, we performed a proteomic analysis in cardiac tissue from a clinically well-phenotyped HCM patient group. METHODS: A proteomics screen was performed in cardiac tissue from 39 HCMSMP patients, 11HCMSMN patients, and 8 nonfailing controls. Patients with HCM had obstructive cardiomyopathy with left ventricular outflow tract obstruction and diastolic dysfunction. A novel MYBPC32373insG mouse model was used to confirm functional relevance of our proteomic findings. RESULTS: In all HCM patient samples, we found lower levels of metabolic pathway proteins and higher levels of extracellular matrix proteins. Levels of t

ADP-ribosyl cyclases du cerveau de souris

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

A la recherche de l'enzyme de détyrosination du C-terminus de l'a-tubuline

Dans les cellules, les microtubules interviennent dans de nombreux événements comme le maintien de l'architecture, la division du matériel génétique, la migration cellulaire ou encore le transport de vésicules et d'organites. Les modifications post-traductionnelles du le C-terminus de la tubuline, bloc de base des microtubules, apparaissent comme très impliquées dans la régulation de ces fonctions car elles régulent le recrutement de leurs nombreux partenaires protéiques. Durant ma thèse, je me suis particulièrement intéressée à une de ces modifications post traductionnelles : le cycle de détyrosination/tyrosination du C-terminus de l'a-tubuline. Ce cycle implique deux enzymes la Tubuline CarboxyPeptidase (TCP), qui clive la tyrosine à l'extrémité de l'a-tubuline, et la Tubuline Tyrosine Ligase (TTL) qui ré-additionne une tyrosine à la tubuline détyrosinée. Des études menées sur ce cycle et notamment la découverte de la TTL ont permis de montrer que la présence d'une tyrosine à l'extrémité C-terminale de l'a-tubuline est indispensable au développement neuronal et que son absence favorise la progression tumorale. La TCP quant à elle est encore inconnue et sa découverte apparaît essentielle afin de pouvoir appréhender le cycle de détyrosination/tyrosination dans sa globalité. Avec pour fil rouge l'identification de la TCP, mon travail s'est déroulé en trois temps. Je me suis tout d'abord intéressée à la protéine EB1. Cette protéine se lie à l'extrémité positive des microtubules où elle recrute de nombreux partenaires microtubulaires. EB1, présente le même C-terminus que l'a-tubuline et notamment une tyrosine terminale indispensable à la liaison des protéines à CAP-Gly. Dans des cellules et des tissus sains j'ai montré qu'EB1 n'existe pas sous forme détyrosinée ce qui souligne la spécificité de la TCP pour l'a-tubuline. Dans un second temps, j'ai participé à l'étude d'une famille de carboxypeptidases cytosoliques impliquées dans la neurodégénérescence, les CCPs, parmi lesquelles nous pensions trouver la TCP. Nous avons montré que quatre de ces enzymes (CCP1, 4, 5, et 6) retirent les glutamates présents de manière latérale au C-terminus de la tubuline. Les CCP1, 4 et 6 peuvent également cliver le dernier glutamate de la tubuline détyrosiné générant de l'a-tubuline où les deux derniers acides aminés ont été clivés (tubuline- 2). Aucune de ces carboxypeptidases ne se révélant être la TCP, j'ai mis en place une méthode biochimique dans le but de purifier cette enzyme. Après plusieurs étapes de purification à partir de cerveaux de souris, des préparations enrichies en activité carboxypeptidase ont été obtenues. Les analyses spectrométriques et bioinformatiques de ces préparations ont permis d'isoler des candidats TCP actuellement testés pour leur potentielle activité de détyrosination du C-terminus de l'a-tubuline. Si la TCP n'est pas présente parmi eux, les outils développés lors de cette étude devraient permettre une très prochaine identification de cette enzyme essentielle.Microtubules control many aspects of cellular function. Those polymers of tubulin are involved in numerous events ranging from the maintenance of cell architecture to cell division and migration through the transport of vesicles and organelles. The post-translational modifications of the C-terminus of tubulin appear to be involved in the regulation of microtubule functions by recruiting different effectors at the growing end of microtubules. During my PhD, I focused on one of these post-translational modifications: the detyrosination/tyrosination cycle of the C-terminal a-tubulin. This cycle involves the enzymatic removal of the C-terminal tyrosine of a-tubulin by an uncharacterized tubulin carboxypeptidase (TCP) and the re-addition of a tyrosine residue by the Tubulin-Tyrosine-Ligase (TTL) isolated in 1975. On one hand, tubulin tyrosination is important in neuronal organization whereas TTL suppression in human cancers is associated with tumor aggressiveness. Those defects are in part due to the failure of microtubule partners to bind detyrosinated microtubules. My project was divided into three main parts. I have first studied EB1, a microtubule plus end tracking protein which recruits many proteins at the microtubule plus end. This protein ends with the same amino acids as does a-tubulin. As in tubulin, the tyrosine terminal is important for the binding of EB1 partners. I showed that EB1 does not exist under detyrosinated form underlying the TCP specificity. Then, I collaborated to identify the function of a carboxypeptidase family within which we thought we could find the TCP. Four members of this family are deglutamylating enzymes (CCP1, CCP4, CCP5 and CCP5). Three of them (CCP1, CCP4 and CCP6) can cleave the last glutamate of detyrosinated tubulin to generate tubulin without the last two C-terminus amino acids ( 2-tubulin). However, none of them was identified as the TCP. I consequently developed a biochemical approach to find this enzyme. Extracts enriched in carboxypeptidase activity after purification steps from mice brain were analyzed by mass spec and bioinformatics. Some candidates are currently tested for their potential C-terminus a-tubulin detyrosinating activity. The tools developed here should allow for pending identification of the TCP.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

[Microtubule polyglutamylation and neurodegeneration].

International audiencen.

La boucle est bouclée: Des enzymes qui détyrosinent les microtubules enfin découvertes

International audienceNo abstract availabl