RESUMO
Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.
Assuntos
Divisão Celular , Microtúbulos , Simbiose , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Microtúbulos/efeitos dos fármacos , Trypanosomatina/genética , Trypanosomatina/metabolismo , Trypanosomatina/ultraestrutura , Trypanosomatina/fisiologia , Ácidos Hidroxâmicos/farmacologia , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Bactérias/metabolismo , Bactérias/genética , Acetilação , Inibidores de Histona Desacetilases/farmacologia , Desacetilase 6 de Histona/metabolismo , Desacetilase 6 de Histona/genética , Citoesqueleto/metabolismo , Citoesqueleto/ultraestruturaRESUMO
Inhibition of histone deacetylase 6 (HDAC6) was shown to support axon growth on the nonpermissive substrates myelin-associated glycoprotein (MAG) and chondroitin sulfate proteoglycans (CSPGs). Though HDAC6 deacetylates α-tubulin, we find that another HDAC6 substrate contributes to this axon growth failure. HDAC6 is known to impact transport of mitochondria, and we show that mitochondria accumulate in distal axons after HDAC6 inhibition. Miro and Milton proteins link mitochondria to motor proteins for axon transport. Exposing neurons to MAG and CSPGs decreases acetylation of Miro1 on Lysine 105 (K105) and decreases axonal mitochondrial transport. HDAC6 inhibition increases acetylated Miro1 in axons, and acetyl-mimetic Miro1 K105Q prevents CSPG-dependent decreases in mitochondrial transport and axon growth. MAG- and CSPG-dependent deacetylation of Miro1 requires RhoA/ROCK activation and downstream intracellular Ca2+ increase, and Miro1 K105Q prevents the decrease in axonal mitochondria seen with activated RhoA and elevated Ca2+ These data point to HDAC6-dependent deacetylation of Miro1 as a mediator of axon growth inhibition through decreased mitochondrial transport.
Assuntos
Desacetilase 6 de Histona/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética , Acetilação/efeitos dos fármacos , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/genética , Cálcio/metabolismo , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Desacetilase 6 de Histona/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Glicoproteína Associada a Mielina/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
HDAC6 is a protein involved in cancer, neurodegenerative disease and inflammatory disorders. To date, the full three-dimensional (3D) structure of human HDAC6 has not been elucidated; however, there are some experimental 3D structural homologs to HDAC6 that can be used as templates. In this work, we utilized molecular modeling procedures to model both of the catalytic domains of HDAC6 connected by the linker region where DMB region is placed. Once the 3D structure of human HDAC6 was obtained, it was structurally evaluated and submitted to docking and molecular dynamic (MD) simulations along with Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method to explore the stability and the binding free energy properties of the HDAC6-ligand complexes. In addition, its structural and energetic behavior was explored with each one of the catalytic domains in the molecular recognition of six selective HDAC6 inhibitors, HPOB, CAY10603, Nexturastat, Rocilinostat, Tubacin and Tubastatin A for DD2, and with the so-called 9-peptide which is DD1-HDAC6 selective substrate. The use of the whole system (DD1-DMB-DD2) showed a tendency toward the ligand affinity of DD2, CAY10603> Tubacin > Rocilinostat > Nexturastat > HPOB > Tubastatin > 9-peptide, which is in line with experimental reports. However, 9-peptide showed a higher affinity for DD1, which agrees with experimental reports elsewhere. Principal component analysis provided important information about the structural changes linked to the molecular recognition process, whereas per-residue decomposition analysis revealed the energetic contribution of the key residues in the molecular binding and structural characteristics that could assist in drug design.