Open Access
MAC and Bcl-2 family proteins conspire in a deadly plot
(Elsevier, 2010-01-18) Dejean, Laurent M.; Ryu, Shin-Young; Martínez-Caballero, Sonia; Teijido Hermida, Óscar; Peixoto, Pablo M.; Kinnally, Kathleen W.; Ciencias de la Salud; Osasun Zientziak
Apoptosis is an elemental form of programmed cell death; it is fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Apoptosis is also involved in many pathologies including cancer, neurodegenerative diseases, aging, and infarcts. This cell death program is tightly regulated by Bcl-2 family proteins by controlling the formation of the mitochondrial apoptosis-induced channel or MAC. Assembly of MAC corresponds to permeabilization of the mitochondrial outer membrane, which is the so called commitment step of apoptosis. MAC provides the pathway through the mitochondrial outer membrane for the release of cytochrome c and other pro-apoptotic factors from the intermembrane space. While overexpression of anti-apoptotic Bcl-2 eliminates MAC activity, oligomers of the pro-apoptotic members Bax and/or Bak are essential structural component(s) of MAC. Assembly of MAC from Bax or Bak was monitored in real time by directly patch-clamping mitochondria with micropipettes containing the sentinel tBid, a direct activator of Bax and Bak. Herein, a variety of high affinity inhibitors of MAC (iMAC) that may prove to be crucial tools in mechanistic studies have recently been identified. This review focuses on characterization of MAC activity, its regulation by Bcl-2 family proteins, and a discussion of how MAC can be pharmacologically turned on or off depending on the pathology to be treated.
Open Access
Pharmacogenomics of Alzheimer's disease: genetic determinants of phenotypic variation and therapeutic outcome
(SciTech Central, 2016-11-16) Cacabelos, Ramón; Carril, Juan Carlos; Cacabelos, Pablo; Teijido Hermida, Óscar; Goldgaber, Dmitry; Ciencias de la Salud; Osasun Zientziak
Alzheimer's disease is a polygenic/complex disorder in which genomic, epigenomic, cerebrovascular, metabolic, and environmental factors converge to define a progressive neurodegenerative phenotype. Conventional anti-dementia drugs are not cost-effective, and pharmacological breakthroughs have not been achieved for the past 10 years. Major determinants of therapeutic outcome in Alzheimer's disease (AD) include age- and sex-related factors, pathogenic phenotype, concomitant disorders, treatment modality and polypharmacy, and pharmacogenetics. Different categories of genes are potentially involved in the pharmacogenetic network responsible for drug efficacy and safety. Pathogenic, mechanistic, metabolic, transporter, and pleiotropic genes represent the major genetic determinants of response to treatment in AD. In pharmacogenetic studies, APOE-4 carriers are the worst responders and APOE-3 carriers are the best responders to conventional treatments. Patients harboring a large (L) number of poly T repeats in intrón 6 of the TOMM40 gene (L/L or S/L genotypes) in haplotypes associated with APOE-4 are the worst responders whereas patients with short (S) TOMM40 poly T variants (S/S genotype), and to a lesser extent S/VL and VL/VL carriers, in haplotypes with APOE-3 are the best responders to treatment. Only 25% of the Caucasian population are extensive metabolizers for trigenic haplotypes integrating CYP2D6- CYP2C19-CYP2C9 variants. Patients harboring CYP-related por (PM) and/or ultra-rapid (UM) geno-phenotypes display more irregular profiles in drug metabolism than extensive (EM) or intermediate (IM) metabolizers. Among 111 pentagenic (APOE-APOB-APOC3-CETP-LPL) haplotypes associated with lipid metabolism, carriers of the H26 haplotype (23-TT-CG- AG-CC) exhibit the lowest cholesterol levels and patients with the H104 haplotype (44-CC-CC-AA-CC) are severely hypercholesterolemic. Epigenetic aberrations (DNA methylation, histone modifications, miRNA dysregulation) in genes configuring the pharmacoepigenetic cascade also influence the response/resistance to drugs. Consequently, novel strategies in drug development, either preventive or therapeutic, for AD should take into consideration these pharmacogenetic determinants for treatment optimization.
Open Access
Current state of theoretical and experimental studies of the voltage-dependent anion channel (VDAC)
(Elsevier, 2016) Noskov, Sergei Y.; Rostovtseva, Tatiana K.; Chamberlin, Adam; Teijido Hermida, Óscar; Jiang, Wei; Bezrukov, Sergey M.; Ciencias de la Salud; Osasun Zientziak
Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane provides a controlled pathway for respiratory metabolites in and out of the mitochondria. In spite of the wealth of experimental data from structural, biochemical, and biophysical investigations, the exact mechanisms governing selective ion and metabolite transport, especially the role of titratable charged residues and interactions with soluble cytosolic proteins, remain hotly debated in the field. The computational advances hold a promise to provide a much sought-after solution to many of the scientific disputes around solute and ion transport through VDAC and hence, across the mitochondrial outer membrane. In this review, we examine how Molecular Dynamics, Free Energy, and Brownian Dynamics simulations of the large β-barrel channel, VDAC, advanced our understanding. We will provide a short overview of non-conventional techniques and also discuss examples of how the modeling excursions into VDAC biophysics prospectively aid experimental efforts.
Open Access
Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel
(American Society for Cell Biology, 2005-03-16) Dejean, Laurent M.; Martínez-Caballero, Sonia; Guo, Liang; Hughes, Cynthia; Teijido Hermida, Óscar; Ducret, Thomas; Ichas, François; Korsmeyer, Stanley J.; Antonsson, Bruno; Jonas, Elizabeth A.; Kinnally, Kathleen W.; Ciencias de la Salud; Osasun Zientziak
Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.
Open Access
Pharmacogenetics of vascular risk factors in Alzheimer's disease
(MDPI, 2018-01-03) Cacabelos, Ramón; Meyyazhagan, Arun; Carril, Juan Carlos; Cacabelos, Pablo; Teijido Hermida, Óscar; Ciencias de la Salud; Osasun Zientziak
Alzheimer's disease (AD) is a polygenic/complex disorder in which genomic, epigenomic, cerebrovascular, metabolic, and environmental factors converge to define a progressive neurodegenerative phenotype. Pharmacogenetics is a major determinant of therapeutic outcome in AD. Different categories of genes are potentially involved in the pharmacogenetic network responsible for drug efficacy and safety, including pathogenic, mechanistic, metabolic, transporter, and pleiotropic genes. However, most drugs exert pleiotropic effects that are promiscuously regulated for different gene products. Only 20% of the Caucasian population are extensive metabolizers for tetragenic haplotypes integrating CYP2D6-CYP2C19-CYP2C9-CYP3A4/5 variants. Patients harboring CYP-related poor (PM) and/or ultra-rapid (UM) geno-phenotypes display more irregular profiles in drug metabolism than extensive (EM) or intermediate (IM) metabolizers. Among 111 pentagenic (APOE-APOB-APOC3-CETP-LPL) haplotypes associated with lipid metabolism, carriers of the H26 haplotype (23-TT-CG-AG-CC) exhibit the lowest cholesterol levels, and patients with the H104 haplotype (44-CC-CC-AA-CC) are severely hypercholesterolemic. Furthermore, APOE, NOS3, ACE, AGT, and CYP variants influence the therapeutic response to hypotensive drugs in AD patients with hypertension. Consequently, the implementation of pharmacogenetic procedures may optimize therapeutics in AD patients under polypharmacy regimes for the treatment of concomitant vascular disorders.
Open Access
Regulation of Bax mitochondrial localization by Bcl-2 and Bcl-xL: keep your friends close but your enemies closer
(Elsevier, 2013) Renault, Thibaud T.; Teijido Hermida, Óscar; Antonsson, Bruno; Dejean, Laurent M.; Manon, Stéphen; Ciencias de la Salud; Osasun Zientziak
Bax-induced mitochondrial outer membrane permeabilization (MOMP) is considered as one of the key control switches of apoptosis. MOMP requires Bax relocation to and insertion into the outer mitochondrial membrane to oligomerize and form pores allowing the release of apoptogenic factors such as cytochrome c. Even if these essential steps are now well-defined, it is necessary to better understand the molecular changes underlying the switch between inactive Bax and active (pore-forming) Bax. One of the ongoing issues is to determine whether Bax mitochondrial translocation is a critical step in the control of Bax activation or if this control is carried by latter regulatory steps. In this focus article we discuss recent data suggesting that although Bcl-2 and Bcl-xL block the MOMP, they can also regulate the mitochondrial Bax content. A new model in which Bax inhibition by Bcl-xL occurs at the immediate proximity of the outer mitochondrial membrane is also discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.
Open Access
Can cloud-based tools accelerate Alzheimer's disease drug discovery?
(Taylor & Francis, 2016) Cacabelos, Ramón; Teijido Hermida, Óscar; Carril, Juan Carlos; Ciencias de la Salud; Osasun Zientziak
Open Access
Vacuolating megalencephalic leukoencephalopathy with subcortical cysts: functional studies of novel variants in MLC1
(Wiley, 2006-02-08) Montagna, Giorgia; Teijido Hermida, Óscar; Eymard-Pierre, Eleonore; Muraki, Koutarou; Cohen, Bruce; Loizzo, Annalivia; Grosso, Pietro; Tedeschi, Gioacchino; Palacín, Manuel; Boespflug-Tanguy, Odile; Bertini, Enrico; Santorelli, Filippo M.; Estévez, Raúl; Ciencias de la Salud; Osasun Zientziak
Nine new unrelated patients presenting vacuolating myelinopathy with subcortical cysts were identified and analyzed for variations in the MLC1 gene. We detected 12 mutations (p.Leu37fs, p.Met80Val, p.Leu83Phe, p.Pro92Ser, p.Ser93Leu, p.Ile108fs, p.Gly130Arg, p.Cys171fs, p.Glu202Lys, p.Ser269Tyr, p.Ala275Asn, and p.Leu310_311insLeu) of which nine were novel. In one patient we did not detect mutations. Using a heterologous system, three new missense variants (p.Glu202Lys, p.Ser269Tyr, and p.Ala275Asn) and a single leucine insertion (p.Leu310insLeu) - lying in a stretch of seven leucines - were functionally assayed by determining total protein levels and mutant protein expression at the plasma membrane. No correlation was observed between mutation, clinical features, and plasma membrane expression of mutant protein.
Open Access
Magic angle spinning nuclear magnetic resonance characterization of voltage-dependent anion channel gating in two-dimensional lipid crystalline bilayers
(American Chemical Society, 2014-12-29) Eddy, Matthew T.; Andreas, Loren; Teijido Hermida, Óscar; Su, Yongchao; Clark, Lindsay; Noskov, Sergei Y.; Wagner, Gerhard; Rostovtseva, Tatiana K.; Griffin, Robert G.; Ciencias de la Salud; Osasun Zientziak
The N-terminus of the voltage-dependent anion channel (VDAC) has been proposed to contain the mechanistically important gating helices that modulate channel opening and closing. In this study, we utilize magic angle spinning nuclear magnetic resonance (MAS NMR) to determine the location and structure of the N-terminus for functional channels in lipid bilayers by measuring long-range 13C–13C distances between residues in the N-terminus and other domains of VDAC reconstituted into DMPC lipid bilayers. Our structural studies show that the distance between A14 Cβ in the N-terminal helix and S193 Cβ is ∼4–6 Å. Furthermore, VDAC phosphorylation by a mitochondrial kinase at residue S193 has been claimed to delay mitochondrial cell death by causing a conformational change that closes the channel, and a VDAC-Ser193Glu mutant has been reported to show properties very similar to those of phosphorylated VDAC in a cellular context. We expressed VDAC-S193E and reconstituted it into DMPC lipid bilayers. Two-dimensional 13C–13C correlation experiments showed chemical shift perturbations for residues located in the N-terminus, indicating possible structural perturbations to that region. However, electrophysiological data recorded on VDAC-S193E showed that channel characteristics were identical to those of wild type samples, indicating that phosphorylation of S193 does not directly affect channel gating. The combination of NMR and electrophysiological results allows us to discuss the validity of proposed gating models.
Open Access
Conductance hysteresis in the voltage-dependent anion channel
(Springer, 2015) Rappaport, Shay M.; Teijido Hermida, Óscar; Hoogerheide, David P.; Rostovtseva, Tatiana K.; Berezhkovskii, Alexander M.; Bezrukov, Sergey M.; Ciencias de la Salud; Osasun Zientziak