Biomedical subjects

Benoit Gilquin

Publications and source records attributed to Benoit Gilquin.

6 recordsLinked to original sources

PubMed2006

HDAC6-p97/VCP controlled polyubiquitin chain turnover.

HDAC6 is a unique cytoplasmic deacetylase capable of interacting with ubiquitin. Using a combination of biophysical, biochemical and biological approaches, we have characterized the ubiquitin-binding domain of HDAC6, named ZnF-UBP, and investigated its biological functions. These studies show that the three Zn ion-containing HDAC6 ZnF-UBP domain presents the highest known affinity for ubiquitin monomers and mediates the ability of HDAC6 to negatively control the cellular polyubiquitin chain turnover. We further show that HDAC6-interacting chaperone, p97/VCP, dissociates the HDAC6-ubiquitin complexes and counteracts the ability of HDAC6 to promote the accumulation of polyubiquitinated proteins. We propose that a finely tuned balance of HDAC6 and p97/VCP concentrations determines the fate of ubiquitinated misfolded proteins: p97/VCP would promote protein degradation and ubiquitin turnover, whereas HDAC6 would favour the accumulation of ubiquitinated protein aggregates and inclusion body formation.

3T3 Cells↗

PubMed2005

Assembly and mechanosensory function of focal adhesions: experiments and models.

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.

Actin Cytoskeleton↗

PubMed2005

Two catalytic domains are required for protein deacetylation.

Histone deacetylase (HDAC)-6 was recently identified as a dual substrate, possibly multisubstrate, deacetylase that can act both on acetylated histone tails and on alpha-tubulin acetylated on Lys40. HDAC-6 is unique among deacetylases in having two hdac domains, and we have used this enzyme as a useful model to dissect the structural requirements for the deacetylation reaction. In this report, we show that both hdac domains are required for the intact deacetylase activity of HDAC-6 in vitro and in vivo. The spatial arrangement of these two domains in HDAC-6 is essential and alteration of the linker region between the two domains severely affects the catalytic activity. Artificial chimeric HDACs, made by replacing the hdac domains in HDAC-6 with corresponding domains from other class II HDACs, show de novo deacetylase activity. Taken together, our results demonstrate for the first time that the spatial arrangement of hdac domains is critical for in vivo deacetylation reaction and may provide a useful model for the development of novel HDAC inhibitors.

3T3 Cells↗

PubMed2005

A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts.

Osteoclast maturation is accompanied by changes in podosome patterning, resulting in the formation of a peripheral belt, which requires an intact microtubule network. Here, we report that by inhibiting Rho, the podosome belt is maintained at the cell periphery despite depolymerisation of microtubules by nocodazole. Rho inhibition was correlated to the increase in microtubule stabilisation and microtubule acetylation. By microinjecting activated Rho or its activated effector mDia2 in osteoclasts, we found that the podosome belt was disrupted and the level of microtubule acetylation dramatically decreased. We further characterised the molecular mechanism responsible for microtubule deacetylation by co-immunoprecipitation experiments. We found that not only was mDia2 coprecipitating with the recently identified microtubule deacetylase HDAC6 but that it also activated the microtubule deacetylase activity of HDAC6 in an in vitro deacetylase assay. Finally, we found that during osteoclastogenesis, there is a correlation between the increase in microtubule acetylation and the podosome belt stabilisation and that if Rho is inhibited in the early stages of osteoclast differentiation, it accelerates both microtubule acetylation and podosome belt stabilisation. Altogether, our data reveal a pathway in which Rho interferes with the osteoclast maturation process by controlling the level of microtubule acetylation and actin organisation through mDIA2 and HDAC6.

Acetylation↗

PubMed2004

Role of the tetradecapeptide repeat domain of human histone deacetylase 6 in cytoplasmic retention.

Histone deacetylase 6 (HDAC6) contains tandem catalytic domains and a ubiquitin-binding zinc finger and displays deacetylase activity toward acetylated microtubules. Here we show that unlike its orthologs from Caenorhabditis elegans, Drosophila, and mouse, human HDAC6 possesses a tetradecapeptide repeat domain located between the second deacetylase domain and the C-terminal ubiquitin-binding motif. Related to this structural difference, the cytoplasmic localization of human, but not murine, HDAC6 is resistant to treatment with leptomycin B (LMB). Although it is dispensable for the deacetylase and ubiquitin binding activities of human HDAC6, the tetradecapeptide repeat domain displays acetyl-microtubule targeting ability. Moreover, it forms a unique structure and is required for the LMB-resistant cytoplasmic localization of human HDAC6. Besides the tetradecapeptide repeat domain, human HDAC6 possesses two LMB-sensitive nuclear export signals and a nuclear localization signal. These results thus indicate that the cytoplasmic localization for murine and human HDAC6 proteins is differentially regulated and suggest that the tetradecapeptide repeat domain serves as an important sequence element to stably retain human HDAC6 in the cytoplasm.

Active Transport, Cell Nucleus↗

PubMed2002

Tat-controlled protein acetylation.

Human immunodeficiency virus, type 1-encoded transactivator protein Tat is known to be a substrate of and to interact with several nuclear histone acetyltransferases (HATs). Here we show that Tat is a general inhibitor of histone acetylation by cellular HATs and that for at least one of them, the CREB-binding protein (CBP), it induces a substrate selectivity. Indeed, in the presence of Tat, the acetylation of histones by CBP was severely inhibited, while that of p53 and MyoD remained unaffected. The C-terminal domain of Tat, dispensable for the activation of viral transcription, was found to be necessary and sufficient to interfere with histone acetylation. These results demonstrate that Tat is able to selectively modulate cellular protein acetylation by nuclear HATs and therefore to take over this specific signaling system in cells.

Acetylation↗