RESUMEN
Biological therapeutic agents are highly targeted and potent but limited in their ability to reach intracellular targets. These limitations often necessitate high therapeutic doses and can be associated with less-than-optimal therapeutic activity. One promising solution for therapeutic agent delivery is use of cell-penetrating peptides. Canonical cell-penetrating peptides, however, are limited by low efficiencies of cellular uptake and endosomal escape, minimal proteolytic stability, and toxicity. To overcome these limitations, we designed a family of proprietary cyclic cell-penetrating peptides that form the core of our endosomal escape vehicle technology capable of delivering therapeutic agent-conjugated cargo intracellularly. We demonstrated the therapeutic potential of this endosomal escape vehicle platform in preclinical models of muscular dystrophy with distinct disease etiology. An endosomal escape vehicle-conjugated, splice-modulating oligonucleotide restored dystrophin protein expression in striated muscles in the mdx mouse, a model for Duchenne muscular dystrophy. Furthermore, another endosomal escape vehicle-conjugated, sterically blocking oligonucleotide led to knockdown of aberrant transcript expression levels in facioscapulohumeral muscular dystrophy patient-derived skeletal muscle cells. These findings suggest a significant therapeutic potential of our endosomal escape vehicle conjugated oligonucleotides for targeted upregulation and downregulation of gene expression in neuromuscular diseases, with possible broader application of this platform for delivery of intracellular biological agents.
RESUMEN
Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Membranas Mitocondriales/fisiología , Animales , Autofagia/fisiología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Restricción Calórica , Células HEK293 , Humanos , Longevidad/fisiología , Masculino , Ratones , Ratones Noqueados , Mitocondrias , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Permeabilidad , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Daño por Reperfusión/metabolismo , Transducción de SeñalRESUMEN
The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle's inner membrane. In mammalian cells the uniporter's activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1-EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2's C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2's function in cells. We propose that in the MICU1-MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.
Asunto(s)
Canales de Calcio/química , Proteínas de Unión al Calcio/química , Proteínas de Transporte de Membrana Mitocondrial/química , Conformación Proteica , Animales , Sitios de Unión , Calcio/química , Canales de Calcio/genética , Proteínas de Unión al Calcio/genética , Cristalografía por Rayos X , Dimerización , Motivos EF Hand/genética , Células HeLa , Humanos , Ratones , Mitocondrias/química , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Estructura Secundaria de ProteínaRESUMEN
The mitochondrial uniporter is a Ca2+-activated Ca2+ channel complex that displays exceptionally high conductance and selectivity. Here, we report cellular metal toxicity screens highlighting the uniporter's role in Mn2+ toxicity. Cells lacking the pore-forming uniporter subunit, MCU, are more resistant to Mn2+ toxicity, while cells lacking the Ca2+-sensing inhibitory subunit, MICU1, are more sensitive than the wild type. Consistent with these findings, Caenorhabditis elegans lacking the uniporter's pore have increased resistance to Mn2+ toxicity. The chemical-genetic interaction between uniporter machinery and Mn2+ toxicity prompted us to hypothesize that Mn2+ can indeed be transported by the uniporter's pore, but this transport is prevented by MICU1. To this end, we demonstrate that, in the absence of MICU1, both Mn2+ and Ca2+ can pass through the uniporter, as evidenced by mitochondrial Mn2+ uptake assays, mitochondrial membrane potential measurements, and mitoplast electrophysiology. We show that Mn2+ does not elicit the conformational change in MICU1 that is physiologically elicited by Ca2+, preventing Mn2+ from inducing the pore opening. Our work showcases a mechanism by which a channel's auxiliary subunit can contribute to its apparent selectivity and, furthermore, may have implications for understanding how manganese contributes to neurodegenerative disease.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Manganeso/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Canales de Calcio/genética , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/genética , Células HEK293 , Humanos , Transporte Iónico/fisiología , Células K562 , Proteínas de Transporte de Membrana Mitocondrial/genéticaRESUMEN
Comparative analyses of transcriptional profiles from humans and mice with cardiovascular pathologies revealed consistently elevated expression of MICU2, a regulatory subunit of the mitochondrial calcium uniporter complex. To determine if MICU2 expression was cardioprotective, we produced and characterized Micu2-/- mice. Mutant mice had left atrial enlargement and Micu2-/- cardiomyocytes had delayed sarcomere relaxation and cytosolic calcium reuptake kinetics, indicating diastolic dysfunction. RNA sequencing (RNA-seq) of Micu2-/- ventricular tissues revealed markedly reduced transcripts encoding the apelin receptor (Micu2-/- vs. wild type, P = 7.8 × 10-40), which suppresses angiotensin II receptor signaling via allosteric transinhibition. We found that Micu2-/- and wild-type mice had comparable basal blood pressures and elevated responses to angiotensin II infusion, but that Micu2-/- mice exhibited systolic dysfunction and 30% lethality from abdominal aortic rupture. Aneurysms and rupture did not occur with norepinephrine-induced hypertension. Aortic tissue from Micu2-/- mice had increased expression of extracellular matrix remodeling genes, while single-cell RNA-seq analyses showed increased expression of genes related to reactive oxygen species, inflammation, and proliferation in fibroblast and smooth muscle cells. We concluded that Micu2-/- mice recapitulate features of diastolic heart disease and define previously unappreciated roles for Micu2 in regulating angiotensin II-mediated hypertensive responses that are critical in protecting the abdominal aorta from injury.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Cardiomiopatía Hipertrófica Familiar/genética , Angiotensina Amida/genética , Angiotensina II/farmacología , Animales , Aorta Abdominal/patología , Canales de Calcio/genética , Proteínas de Unión al Calcio/genética , Cardiomiopatía Hipertrófica Familiar/patología , Electrocardiografía , Regulación de la Expresión Génica , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Ratones Endogámicos C57BL , Ratones Mutantes , Mitocondrias Hepáticas/fisiología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiologíaRESUMEN
The mitochondrial calcium uniporter is a Ca2+-activated Ca2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca2+ signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca2+ We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca2+-binding affinity of MICU1-MICU2. We conclude that cooperative, high-affinity interaction of the MICU1-MICU2 complex with Ca2+ serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca2+ signals.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Cardiolipinas/metabolismo , Citosol/metabolismo , Motivos EF Hand/fisiología , Células HEK293 , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismoRESUMEN
The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of â¼20-60 amino acids, a net charge between +3 to +6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, â¼80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Proteómica/métodos , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Evolución Molecular , Humanos , Riñón/metabolismo , Hígado/metabolismo , Ratones , Mitocondrias/genética , Proteínas Mitocondriales/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
OBJECTIVE: To define the mechanism responsible for fatigue, lethargy, and weakness in 2 cousins who had a normal muscle biopsy. METHODS: Exome sequencing, long-range PCR, and Sanger sequencing to identify the pathogenic mutation. Functional analysis in the patient fibroblasts included oxygen consumption measurements, extracellular acidification studies, Western blotting, and calcium imaging, followed by overexpression of the wild-type protein. RESULTS: Analysis of the exome sequencing depth revealed a homozygous deletion of exon 1 of MICU1 within a 2,755-base pair deletion. No MICU1 protein was detected in patient fibroblasts, which had impaired mitochondrial calcium uptake that was rescued through the overexpression of the wild-type allele. CONCLUSIONS: MICU1 mutations cause fatigue and lethargy in patients with normal mitochondrial enzyme activities in muscle. The fluctuating clinical course is likely mediated through the mitochondrial calcium uniporter, which is regulated by MICU1.
RESUMEN
The mitochondrial calcium uniporter is an evolutionarily conserved calcium channel, and its biophysical properties and relevance to cell death, bioenergetics and signalling have been investigated for decades. However, the genes encoding this channel have only recently been discovered, opening up a new 'molecular era' in the study of its biology. We now know that the uniporter is not a single protein but rather a macromolecular complex consisting of pore-forming and regulatory subunits. We review recent studies that harnessed the power of molecular biology and genetics to characterize the mechanism of action of the uniporter, its evolution and its contribution to physiology and human disease.
Asunto(s)
Canales de Calcio/fisiología , Animales , Canales de Calcio/química , Señalización del Calcio , Retroalimentación Fisiológica , Humanos , Modelos MolecularesRESUMEN
Installing an electrophilic amino-acid residue can engender a peptide or protein with chemoselective reactivity. Such a modification to collagen, which is the most abundant protein in animals, could facilitate the development of new biomaterials. Collagen has an abundance of proline-like residues. Here, we report on the incorporation of an electrophilic proline congener, (2S)-4-ketoproline (Kep), into a collagen-mimetic peptide (CMP). An ab initio conformational analysis of Kep revealed its potential to be accommodated within a collagen triple helix. A synthetic CMP containing a Kep residue was indeed able to form a stable triple helix. Moreover, the condensation of its carbonyl group with aminooxy-biotin did not compromise the conformational stability of the triple helix. These data encourage the use of 4-ketoproline as an electrophilic congener of proline.
Asunto(s)
Prolina/análogos & derivados , Colágeno/química , Péptidos/química , Prolina/química , Conformación ProteicaRESUMEN
APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.
Asunto(s)
Ascorbato Peroxidasas/biosíntesis , Microscopía Electrónica de Transmisión/métodos , Proteómica/métodos , Animales , Ascorbato Peroxidasas/genética , Células COS , Proteínas de Unión al Calcio/análisis , Proteínas de Transporte de Catión/análisis , Chlorocebus aethiops , Evolución Molecular Dirigida/métodos , Células HEK293 , Células HeLa , Humanos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/análisis , Saccharomyces cerevisiae/enzimologíaRESUMEN
The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits--mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)--and two soluble regulatory components--MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.
Asunto(s)
Canales de Calcio/química , Calcio/química , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Calcio/metabolismo , Línea Celular , Dictyostelium , Técnicas Genéticas , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismoAsunto(s)
Canales de Calcio/fisiología , Proteínas de Unión al Calcio/fisiología , Calcio/metabolismo , Proteínas de Transporte de Catión/fisiología , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Canales de Calcio/química , Células HEK293 , Humanos , Proteínas Mitocondriales/fisiologíaRESUMEN
The mitochondrial uniporter is a selective Ca(2+) channel regulated by MICU1, an EF hand-containing protein in the organelle's intermembrane space. MICU1 physically associates with and is co-expressed with a paralog, MICU2. To clarify the function of MICU1 and its relationship to MICU2, we used gene knockout (KO) technology. We report that HEK-293T cells lacking MICU1 or MICU2 lose a normal threshold for Ca(2+) intake, extending the known gating function of MICU1 to MICU2. Expression of MICU1 or MICU2 mutants lacking functional Ca(2+)-binding sites leads to a striking loss of Ca(2+) uptake, suggesting that MICU1/2 disinhibit the channel in response to a threshold rise in [Ca(2+)]. MICU2's activity and physical association with the pore require the presence of MICU1, though the converse is not true. We conclude that MICU1 and MICU2 are nonredundant and together set the [Ca(2+)] threshold for uniporter activity.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Sitios de Unión , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Células HEK293 , Humanos , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , MutaciónRESUMEN
The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its molecular components include the EF-hand-containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex). Quantitative mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and essential MCU regulator (EMRE), a previously uncharacterized protein. EMRE is a 10-kilodalton, metazoan-specific protein with a single transmembrane domain. In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and additionally bridges the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Secuencia de Aminoácidos , Canales de Calcio/química , Canales de Calcio/genética , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/genética , Motivos EF Hand , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteínas de Transporte de Membrana Mitocondrial/genética , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , ProteómicaRESUMEN
Carbonyl-carbonyl (C=O···C'=O') interactions are ubiquitous in both small and large molecular systems. This interaction involves delocalization of a lone pair (n) of a donor oxygen into the antibonding orbital (π*) of an acceptor carbonyl group. Analyses of high-resolution protein structures suggest that these carbonyl-carbonyl interactions prefer to occur in pairs, that is, one donor per acceptor. Here, the reluctance of the acceptor carbonyl group (C'=O') to engage in more than one nâπ* electron delocalization is probed using imidazolidine-based model systems with one acceptor carbonyl group and two equivalent donor carbonyl groups. The data indicate that the electrophilicity of the acceptor carbonyl group is reduced when it engages in nâπ* electron delocalization. This diminished electrophilicity discourages a second nâπ* interaction with the acceptor carbonyl group.
Asunto(s)
Ésteres/química , Electrones , Imidazolidinas/química , Modelos Moleculares , Estructura Molecular , Oxidación-ReducciónRESUMEN
Mitochondrial Ca(2+) uptake via the uniporter is central to cell metabolism, signaling, and survival. Recent studies identified MCU as the uniporter's likely pore and MICU1, an EF-hand protein, as its critical regulator. How this complex decodes dynamic cytoplasmic [Ca(2+)] ([Ca(2+)]c) signals, to tune out small [Ca(2+)]c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca(2+) accumulation during small [Ca(2+)]c elevations but an attenuated response to agonist-induced [Ca(2+)]c pulses. The latter reflects loss of positive cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca(2+)]c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca(2+) binding, yet cells show impaired oxidative metabolism and sensitization to Ca(2+) overload. Collectively, the data indicate that MICU1 senses the [Ca(2+)]c to establish the uniporter's threshold and gain, thereby allowing mitochondria to properly decode different inputs.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Células Cultivadas , Células HEK293 , Células HeLa , Hepatocitos/metabolismo , Humanos , Ratones , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Complejos Multiproteicos/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Canales de Calcio/genética , Señalización del Calcio , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Respiración de la Célula/genética , Células HEK293 , Células HeLa , Humanos , Hígado/metabolismo , Potencial de la Membrana Mitocondrial/genética , Ratones , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Familia de Multigenes , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Interferencia de ARN , Alineación de SecuenciaRESUMEN
Amide carbonyl groups in proteins can engage in CâO···CâO and C-X···CâO interactions, where X is a halogen. The putative involvement of four poles suggests that these interactions are primarily dipolar. Our survey of crystal structures with a C-X···CâO contact that is short (i.e., within the sum of the X and C van der Waals radii) revealed no preferred C-X···CâO dihedral angle. Moreover, we found that structures with a short X(-)···CâO contact display the signatures of an nâπ* interaction. We conclude that intimate interactions with carbonyl groups do not require a dipole.
Asunto(s)
Amidas/química , Halógenos/química , Hidrocarburos Halogenados/química , Proteínas/química , Cristalografía por Rayos X , Modelos Moleculares , Carbonilación Proteica , TermodinámicaRESUMEN
The chromophore of fluorescent proteins, including the green fluorescent protein (GFP), contains a highly conjugated imidazolidinone ring. In many fluorescent proteins, the carbonyl group of the imidazolidinone ring engages in a hydrogen bond with the side chain of an arginine residue. Prior studies have indicated that such an electrophilic carbonyl group in a protein often accepts electron density from a main-chain oxygen. A survey of high-resolution structures of fluorescent proteins indicates that electron lone pairs of a main-chain oxygen-Thr62 in GFP-donate electron density into an antibonding orbital of the imidazolidinone carbonyl group. This nâπ* electron delocalization prevents structural distortion during chromophore excitation that could otherwise lead to fluorescence quenching. In addition, this interaction is present in on-pathway intermediates leading to the chromophore, and thus could direct its biogenesis. Accordingly, this nâπ* interaction merits inclusion in computational and photophysical analyses of the chromophore, and in speculations about the molecular evolution of fluorescent proteins.