RESUMO
The oxysterol-binding protein-related proteins (ORPs) have emerged as orchestrators of phosphatidylinositol-4,5-bisphosphate (PIP2) and cholesterol trafficking to the plasma membrane (PM). In this scenario, recent studies raised the prospect of ORPs cooperative behavior in sustaining leukemia stem cells (LSCs) survival by remotely enhancing ER-mitochondria Ca2+ communication. At the apex of the signaling cascade, the aberrantly upregulated LSC-ORP4L fosters PM-PIP2 extraction & cleavage, endoplasmic reticulum (ER)-Ca2+ release and mitochondrial energetics. The theoretical ember of draining fuel from the chemoresistant LSCs by restraining endoplasmic reticulum (ER)-mitochondria Ca2+ fluxes in a lipid-contingent fashion ensues. In light of relevant literature, this review briefly and critically discusses some key molecular ins & outs underlying such therapeutic opportunity in acute myeloid leukemia (AML).
Assuntos
Cálcio/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Colesterol/metabolismo , Humanos , Transdução de SinaisRESUMO
The pro- and antiapoptotic proteins belonging to the B-cell lymphoma-2 (Bcl-2) family exert a critical control over cell-death processes by enabling or counteracting mitochondrial outer membrane permeabilization. Beyond this mitochondrial function, several Bcl-2 family members have emerged as critical modulators of intracellular Ca2+ homeostasis and dynamics, showing proapoptotic and antiapoptotic functions. Bcl-2 family proteins specifically target several intracellular Ca2+-transport systems, including organellar Ca2+ channels: inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), Ca2+-release channels mediating Ca2+ flux from the endoplasmic reticulum, as well as voltage-dependent anion channels (VDACs), which mediate Ca2+ flux across the mitochondrial outer membrane into the mitochondria. Although the formation of protein complexes between Bcl-2 proteins and these channels has been extensively studied, a major advance during recent years has been elucidating the complex interaction of Bcl-2 proteins with IP3Rs. Distinct interaction sites for different Bcl-2 family members were identified in the primary structure of IP3Rs. The unique molecular profiles of these Bcl-2 proteins may account for their distinct functional outcomes when bound to IP3Rs. Furthermore, Bcl-2 inhibitors used in cancer therapy may affect IP3R function as part of their proapoptotic effect and/or as an adverse effect in healthy cells.
Assuntos
Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Homeostase , Humanos , Camundongos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Domínios Proteicos , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Proteína bcl-X/metabolismoRESUMO
Intracellular Ca2+-flux systems located at the ER-mitochondrial axis govern mitochondrial Ca2+ balance and cell fate. Multiple yet incurable pathologies are characterized by insufficient or excessive Ca2+ fluxes toward the mitochondria, in turn leading to aberrant cell life or death dynamics. The discovery and ongoing molecular characterization of the main interorganellar Ca2+ gateways have resulted in a novel class of peptide tools able to regulate relevant protein-protein interactions (PPIs) underlying this signaling scenario. Here, we review peptides, molecularly derived from Ca2+-flux systems or their accessory proteins. We discuss how they alter Ca2+-signaling protein complexes and modulate cell survival in light of their forthcoming therapeutic applications.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Peptídeos/uso terapêuticoRESUMO
Sarcopenia is the age-related loss of skeletal muscle mass, strength, and function. It is associated with regenerative difficulties by satellite cells, adult muscle stem cells, and alteration of oxidative management, mainly the increase in superoxide anions (O2â¢-). We aimed to investigate the relation between regenerative deficit in elderly and increase in O2â¢- production along with mitochondrial alterations. Myoblasts and myotubes from skeletal muscle of young and elderly healthy subjects (27.8 ± 6 and 72.4 ± 6.5 years old) were measured: (1) superoxide dismutase activity and protein content, (2) mitochondrial O2â¢- production levels, (3) O2â¢- production variability, and (4) mitochondrial bioenergetic profile. Compared to young myoblasts, elderly myoblasts displayed decreased SOD2 protein expression, elevated mitochondrial O2â¢- baseline levels, and decreased oxidative phosphorylation and glycolysis. Additionally, elderly versus young myotubes showed elevated mitochondrial O2â¢- levels when stressed with N-acetyl cysteine or high glucose and higher glycolysis despite showing comparable oxidative phosphorylation levels. Altogether, the elderly may have less metabolic plasticity due to the impaired mitochondrial function caused by O2â¢-. However, the increased energy demand related to the differentiation process appears to activate compensatory mechanisms for the partial mitochondrial dysfunction.
Assuntos
Biópsia/métodos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Superóxidos/metabolismo , Adulto , Idoso , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Masculino , Mioblastos/patologiaRESUMO
B-cell lymphoma 2 (Bcl-2) protein is the archetype apoptosis suppressor protein. The N-terminal Bcl-2-homology 4 (BH4) domain of Bcl-2 is required for the antiapoptotic function of this protein at the mitochondria and endoplasmic reticulum (ER). The involvement of the BH4 domain in Bcl-2's antiapoptotic functions has been proposed based on Gly-based substitutions of the Ile14/Val15 amino acids, two hydrophobic residues located in the center of Bcl-2's BH4 domain. Following this strategy, we recently showed that a BH4-domain-derived peptide in which Ile14 and Val15 have been replaced by Gly residues, was unable to dampen proapoptotic Ca2+ -release events from the ER. Here, we investigated the impact of these mutations on the overall structure, stability, and function of full-length Bcl-2 as a regulator of Ca2+ signaling and cell death. Our results indicate that full-length Bcl-2 Ile14Gly/Val15Gly, in contrast to wild-type Bcl-2, (a) displayed severely reduced structural stability and a shortened protein half-life; (b) failed to interact with Bcl-2-associated X protein (BAX), to inhibit the inositol 1,4,5-trisphosphate receptor (IP3 R) and to protect against Ca2+ -mediated apoptosis. We conclude that the hydrophobic face of Bcl-2's BH4 domain (Ile14, Val15) is an important structural regulatory element by affecting protein stability and turnover, thereby likely reducing Bcl-2's ability to modulate the function of its targets, like IP3 R and BAX. Therefore, Bcl-2 structure/function studies require pre-emptive and reliable determination of protein stability upon introduction of point mutations at the level of the BH4 domain.
Assuntos
Isoleucina/genética , Mutação Puntual , Proteínas Proto-Oncogênicas c-bcl-2/genética , Valina/genética , Animais , Apoptose/genética , Células COS , Cálcio/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Isoleucina/química , Isoleucina/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Valina/química , Valina/metabolismoRESUMO
Mitochondria are important regulators of cell death and cell survival. Mitochondrial Ca2+ levels are critically involved in both of these processes. On the one hand, excessive mitochondrial Ca2+ leads to Ca2+-induced mitochondrial outer membrane permeabilization and thus apoptosis. On the other hand, mitochondria need Ca2+ in order to efficiently fuel the tricarboxylic acid cycle and maintain adequate mitochondrial bioenergetics. For obtaining this Ca2+, the mitochondria are largely dependent on close contact sites with the endoplasmic reticulum (ER), the so-called mitochondria-associated ER membranes. There, the inositol 1,4,5-trisphosphate receptors are responsible for the Ca2+ release from the ER. It comes as no surprise that this Ca2+ release from the ER and the subsequent Ca2+ uptake at the mitochondria are finely regulated. Cancer cells often modulate ER-Ca2+ transfer to the mitochondria in order to promote cell survival and to inhibit cell death. Important regulators of these Ca2+ signals and the onset of cancer are the B-cell lymphoma 2 (Bcl-2) family of proteins. An increasing number of reports highlight the ability of these Bcl-2-protein family members to finely regulate Ca2+ transfer from ER to mitochondria both in healthy cells and in cancer. In this review, we focus on recent insights into the dynamic regulation of ER-mitochondrial Ca2+ fluxes by Bcl-2-family members and how this impacts cell survival, cell death and mitochondrial energy production.
RESUMO
BACKGROUND: Alkylated DNA-protein alkyltransferases (AGTs) are conserved proteins that repair alkylation damage in DNA by using a single-step mechanism leading to irreversible alkylation of the catalytic cysteine in the active site. Trans-alkylation induces inactivation and destabilization of the protein, both in vitro and in vivo, likely triggering conformational changes. A complete picture of structural rearrangements occurring during the reaction cycle is missing, despite considerable interest raised by the peculiarity of AGT reaction, and the contribution of a functional AGT in limiting the efficacy of chemotherapy with alkylating drugs. METHODS: As a model for AGTs we have used a thermostable ortholog from the archaeon Sulfolobus solfataricus (SsOGT), performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNA-bound and mutated versions of the protein. RESULTS: Conformational changes occurring during lesion recognition and after the reaction, allowed us to identify a novel interaction network contributing to SsOGT stability, which is perturbed when a bulky adduct between the catalytic cysteine and the alkyl group is formed, a mandatory step toward the permanent protein alkylation. CONCLUSIONS: Our data highlighted conformational changes and perturbation of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N- and C-terminal domains of SsOGT is important for protein activity and stability. GENERAL SIGNIFICANCE: A general model of structural rearrangements occurring during the reaction cycle of AGTs is proposed. If confirmed, this model might be a starting point to design strategies to modulate AGT activity in therapeutic settings.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Alquilantes/metabolismo , Alquilação/fisiologia , Catálise , Reparo do DNA/fisiologia , Domínios Proteicos , Estabilidade Proteica , Sulfolobus solfataricus/metabolismoRESUMO
The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2ß, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/química , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cálcio/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Receptores de Inositol 1,4,5-Trifosfato/química , Domínios Proteicos , Sulfonamidas/farmacologiaRESUMO
Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca(2+)-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), while Bcl-XL, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-XL co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-XL to bind to RyRs, loading the BH4-Bcl-XL peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca(2+) release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-XL, the Bcl-XL(K87D) mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca(2+) release. These data suggest that Bcl-XL binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteína bcl-X/metabolismo , Cálcio/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Proteína bcl-X/química , Proteína bcl-X/genéticaRESUMO
Reference treatment of advanced prostate cancer (PCa) relies on pharmacological or surgical androgen deprivation therapy. However, it is only temporarily efficient as tumor cells inevitably adapt to the low testosterone environment and become hormone-refractory (HRPCa). We observed that androgen removal in HRPCa-derived LNCaP cells causes different alterations in their Ca(2+) homeostasis among which a reduction of ER Ca(2+) content. We show that the decrease in [Ca(2+)]ER is due to a modest overexpression of type 1 IP3R and a threefold increased phosphorylation of IP3R1 on Ser-1716, a protein kinase A (PKA) consensus site, both implicated in ER Ca(2+) leak. Accordingly, ER Ca(2+) content was restored by siRNA-mediated down-regulation of IP3R1 or by inhibition of its phosphorylation by competition with a permeant TAT-peptide containing the Ser-1716 consensus phosphorylation sequence or by treatment with the PKA inhibitor H89. Moreover, inhibition of the IP3R1 phosphorylation by both methods sensitized the LNCaP cells to androgen deprivation-induced apoptosis. In addition, SERCA2b overexpression precluded the effect of androgen deprivation on ER Ca(2+) store content and reduced resistance to androgen deprivation. Taken together, these results indicate that lowering the ER Ca(2+)-store content by increasing IP3R1 levels and IP3R1 phosphorylation by PKA is a protective mechanism by which HRPCa-derived cells escape cell death in the absence of androgenic stimulation.
Assuntos
Androgênios/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Isoquinolinas/farmacologia , Masculino , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sulfonamidas/farmacologia , Testosterona/metabolismo , Transgenes/genética , Evasão TumoralRESUMO
Excessive Ca(2+) fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca(2+)-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca(2+)-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca(2+) uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1(-/-) cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca(2+) uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca(2+) release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca(2+) uptake into the mitochondria.
Assuntos
Apoptose , Sinalização do Cálcio , Mitocôndrias/metabolismo , Canal de Ânion 1 Dependente de Voltagem/fisiologia , Proteína bcl-X/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Camundongos , Dados de Sequência MolecularRESUMO
The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca(2+)-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+) release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Permeabilidade/efeitos dos fármacos , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaAssuntos
Benzopiranos/farmacologia , Compostos de Bifenilo/farmacologia , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Nitrilas/farmacologia , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Células HEK293 , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tapsigargina/farmacologiaRESUMO
Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca(2+) signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP3R): Bcl-2 likely acts as an endogenous inhibitor of the IP3R, preventing pro-apoptotic Ca(2+) transients, while Bcl-XL likely acts as an endogenous IP3R-sensitizing protein promoting pro-survival Ca(2+) oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-XL may underlie the divergence in IP3R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP3R, is likely to be Bcl-2's determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca(2+)-channel tail and might be more crucial for Bcl-XL's function. Furthermore, one amino acid critically different in the sequence of Bcl-2's and Bcl-XL's BH4 domains underpins their selective effect on Ca(2+) signaling and distinct biological properties of Bcl-2 versus Bcl-XL. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-XL functions in normal cells. Thus, IP3R-derived molecules that mimic the BH4 domain's binding site on the IP3R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2's proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP3Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Modelos Biológicos , Família Multigênica/genética , Família Multigênica/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Especificidade por SubstratoRESUMO
Several members of the anti-apoptotic Bcl-2-protein family, including Bcl-2, Bcl-X(L) and Mcl-1, directly bind and regulate the inositol 1,4,5-trisphosphate receptor (IP(3)R), one of the two main intracellular Ca(2+)-release channel types present in the endoplasmic reticulum. However, the molecular determinants underlying their binding to the IP(3)R remained a matter of debate. One interaction site for Bcl-2 was proposed in the central part of the modulatory domain [Y.P. Rong, A.S. Aromolaran, G. Bultynck, F. Zhong, X. Li, K. McColl, S. Matsuyama, S. Herlitze, H.L. Roderick, M.D. Bootman, G.A. Mignery, J.B. Parys, H. De Smedt, C.W. Distelhorst, Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals, Mol. Cell 31 (2008) 255-265] and another site in the C-terminal domain of the IP(3)R encompassing the sixth transmembrane domain, to which Bcl-2, Bcl-X(L) and Mcl-1 can bind [E.F. Eckenrode, J. Yang, G.V. Velmurugan, J.K. Foskett, C. White, Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca(2+) signaling, J. Biol. Chem. 285 (2010) 13678-13684]. Here, we investigated and compared the binding of Bcl-2 and Bcl-X(L) to both sites. Two different IP(3)R domains were used for the C-terminal site: one lacking and one containing the sixth transmembrane domain. Our results show that elements preceding the C-terminal cytosolic tail located at the sixth transmembrane domain of IP(3)R1 were critical for recruiting both Bcl-2 and Bcl-X(L) to the C-terminal part of the IP(3)R. Furthermore, consistent with our previous observations, Bcl-X(L) bound with higher efficiency to the C-terminal part of the IP(3)R and to a much lesser extent to the central, modulatory domain, while Bcl-2 targeted both sites with similar efficiencies. In conclusion, IP(3)R harbors two different binding sites for anti-apoptotic Bcl-2 proteins, one in the central, modulatory domain and one in the C-terminal domain near the Ca(2+)-channel pore.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Clonagem Molecular , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Camundongos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMO
Bcl-2 contributes to the pathophysiology and therapeutic resistance of chronic lymphocytic leukemia (CLL). Therefore, developing inhibitors of this protein based on a thorough understanding of its mechanism of action is an active and promising area of inquiry. One approach centers on agents (eg, ABT-737) that compete with proapoptotic members of the Bcl-2 protein family for binding in the hydrophobic groove formed by the BH1-BH3 domains of Bcl-2. Another region of Bcl-2, the BH4 domain, also contributes to the antiapoptotic activity of Bcl-2 by binding to the inositol 1,4,5-trisphosphate receptor (IP3R) Ca²(+) channel, inhibiting IP(3)-dependent Ca²(+) release from the endoplasmic reticulum. We report that a novel synthetic peptide, modeled after the Bcl-2-interacting site on the IP3R, binds to the BH4 domain of Bcl-2 and functions as a competitive inhibitor of the Bcl-2-IP3R interaction. By disrupting the Bcl-2-IP3R interaction, this peptide induces an IP3R-dependent Ca²(+) elevation in lymphoma and leukemia cell lines and in primary CLL cells. The Ca²(+) elevation evoked by this peptide induces apoptosis in CLL cells, but not in normal peripheral blood lymphocytes, suggesting the involvement of the Bcl-2-IP3R interaction in the molecular mechanism of CLL and indicating the potential merit of targeting this interaction therapeutically.
Assuntos
Apoptose/fisiologia , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligação Competitiva , Western Blotting , Linhagem Celular Tumoral , Humanos , ImunoprecipitaçãoRESUMO
BACKGROUND: The loss of cell cycle regulation due to abnormal function of cyclin-dependent kinases (cdk) occurs in tumors and leads to genetic instability of chemotherapy-resistant cells. In this study, we investigated the effect of the cdk inhibitor flavopiridol in anaplastic large cell lymphomas, in which unrestrained proliferation depends on NPM-ALK tyrosine kinase activity. DESIGN AND METHODS: Effects of flavopiridol were examined in ALK-positive and -negative anaplastic large cell lymphoma cells by means of immunoblotting and immunofluorescence analyses to assess cdk expression and activity, quantitative real time reverse transcriptase polymerase chain reaction to measure drug-induced changes in transcription, and FACS analyses to monitor changes in proliferation and survival. RESULTS: Treatment with flavopiridol resulted in growth inhibition of anaplastic large cell lymphoma cells, along with accumulation of subG(1) cells and disappearance of S phase without cell cycle arrest. Consistent with flavopiridol activity, phosphorylation at cdk2, cdk4, cdk9 sites on RB and RNA polymerase II was inhibited. This correlated with induction of cell death through rapid mitochondrial damage, inhibition of DNA synthesis, and down-regulation of anti-apoptotic proteins and transcripts. Notably, flavopiridol was less active in ALK-positive cells, as apoptosis was observed at higher concentrations and later time points, and resistance to treatment was observed in cells maintaining NPM-ALK signaling. NPM-ALK inhibition affected proliferation but not survival of anaplastic large cell lym-phoma cells, whereas it resulted in a dramatic increase in apoptosis when combined with flavopiridol. CONCLUSIONS: This work provides the first demonstration that targeting cdk is effective against anaplastic large cell lymphoma cells, and proves the critical role of NPM-ALK in the regulation of responsiveness of tumor cells with cdk dysregulation.
Assuntos
Antineoplásicos/farmacologia , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Piperidinas/farmacologia , Proteínas Tirosina Quinases/biossíntese , Apoptose , Bromodesoxiuridina/farmacologia , Ciclo Celular , Separação Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Fatores de TempoRESUMO
A distinctive feature in the topographic organization of the olfactory system in mammals is the dual function of the odorant receptor (OR): it detects odors in the nasal epithelium and plays an instructive role in the axonal convergence of olfactory sensory neurons (OSN) into the olfactory bulb (OB). The latter function is supported by genetic experiments and by the expression of the OR not only on the cilia, but also on the axon termini of the OSN. The signaling pathway coupled to the OR on the cilia is well known and is recognized to involve cAMP and Ca(2+), whereas, until now, nothing was known on the functional characteristics of the OR on the axon termini-growth cone. Here, by analyzing the spatiotemporal dynamics of cAMP and Ca(2+) in living OSN in vitro and in situ, we found that the OR at the growth cone is capable of binding odors and is coupled to cAMP synthesis and Ca(2+) influx through cyclic nucleotide gated (CNG) channels. Furthermore we found that selective odor activation of the OR on the growth cone is followed by nuclear translocation of protein kinase A catalytic subunit. These results define the functional properties of the OR on the growth cone and suggest a potential role of OR activation in axonal convergence and sensory map formation.