RESUMEN
Most available information on endoplasmic reticulum (ER)-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca(2+) entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here, we show that the three extended synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca(2+) in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca(2+) regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca(2+) entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast) mediates the formation of ER-PM contacts sites, which are functionally distinct from those mediated by STIM1 and Orai1.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinaptotagminas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestructura , Retículo Endoplásmico/química , Retículo Endoplásmico/ultraestructura , Células HeLa , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Sinaptotagminas/química , Sinaptotagminas/genética , Levaduras/citología , Levaduras/metabolismoRESUMEN
The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Sensoras del Calcio Intracelular/metabolismo , Proteínas Musculares/metabolismo , Selenoproteínas/metabolismo , Células HeLa , Humanos , Proteínas Sensoras del Calcio Intracelular/genética , Proteínas Musculares/genética , Oxidación-Reducción , Selenoproteínas/genéticaRESUMEN
The tryptophan rich basic protein/calcium signal-modulating cyclophilin ligand (WRB/CAML) and Get1p/Get2p complexes, in vertebrates and yeast, respectively, mediate the final step of tail-anchored protein insertion into the endoplasmic reticulum membrane via the Get pathway. While WRB appears to exist in all eukaryotes, CAML homologs were previously recognized only among chordates, raising the question as to how CAML's function is performed in other phyla. Furthermore, whereas WRB was recognized as the metazoan homolog of Get1, CAML and Get2, although functionally equivalent, were not considered to be homologous. CAML contains an N-terminal basic, TRC40/Get3-interacting, region, three transmembrane segments near the C-terminus, and a poorly conserved region between these domains. Here, I searched the NCBI protein database for remote CAML homologs in all eukaryotes, using position-specific iterated-basic local alignment search tool, with the C-terminal, the N-terminal or the full-length sequence of human CAML as query. The N-terminal basic region and full-length CAML retrieved homologs among metazoa, plants and fungi. In the latter group several hits were annotated as GET2. The C-terminal query did not return entries outside of the animal kingdom, but did retrieve over one hundred invertebrate metazoan CAML-like proteins, which all conserved the N-terminal TRC40-binding domain. The results indicate that CAML homologs exist throughout the eukaryotic domain of life, and suggest that metazoan CAML and yeast GET2 share a common evolutionary origin. They further reveal a tight link between the particular features of the metazoan membrane-anchoring domain and the TRC40-interacting region. The list of sequences presented here should provide a useful resource for future studies addressing structure-function relationships in CAML proteins.
Asunto(s)
Retículo Endoplásmico , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Retículo Endoplásmico/metabolismo , Neuronas Motoras/metabolismo , Neuritas/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Neuronas Motoras/patología , Mutación , Neuritas/patología , Ratas , Proteínas de Transporte Vesicular/genéticaRESUMEN
Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.
Asunto(s)
Citocromos b5/metabolismo , Retículo Endoplásmico/metabolismo , Membranas Mitocondriales/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Citocromos b5/química , Células HeLa , Humanos , Dominios Proteicos , Transporte de Proteínas , Proteínas R-SNARE/metabolismoRESUMEN
Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/metabolismo , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Retículo Endoplásmico/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.
Asunto(s)
Subunidades de Proteína/metabolismo , Receptores Nicotínicos/metabolismo , Sitios de Unión/fisiología , Membrana Celular/metabolismo , Células Cultivadas , HumanosRESUMEN
The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward-bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi-to-plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal-deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail-anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy- and Rab6-dependent, and Rab6 inhibition accelerated signal-deleted VSVG's transport to the cell surface. Our results extend the dynamic bi-directional relationship between the Golgi and ER to include surface-directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Vesículas Secretoras/metabolismo , Animales , Línea Celular , Glicoproteínas/metabolismo , Humanos , Ratas , Proteínas Virales/metabolismoRESUMEN
Secretory proteins exit the endoplasmic reticulum (ER) in coat protein complex II (COPII)-coated vesicles and then progress through the Golgi complex before delivery to their final destination. Soluble cargo can be recruited to ER exit sites by signal-mediated processes (cargo capture) or by bulk flow. For membrane proteins, a third mechanism, based on the interaction of their transmembrane domain (TMD) with lipid microdomains, must also be considered. In this Commentary, I review evidence in favor of the idea that partitioning of TMDs into bilayer domains that are endowed with distinct physico-chemical properties plays a pivotal role in the transport of membrane proteins within the early secretory pathway. The combination of such self-organizational phenomena with canonical intermolecular interactions is most likely to control the release of membrane proteins from the ER into the secretory pathway.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Humanos , Transporte de Proteínas , Vías SecretorasRESUMEN
The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in â¼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.
Asunto(s)
ATPasas Transportadoras de Arsenitos/química , ATPasas Transportadoras de Arsenitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Animales , ATPasas Transportadoras de Arsenitos/genética , Línea Celular , Células Cultivadas , Síndrome de Down/genética , Síndrome de Down/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Microsomas Hepáticos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades de Proteína , Transporte de Proteínas , Proteolípidos/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Heteromeric nAChRs are pentameric cation channels, composed of combinations of two or three α and three or two ß subunits, which play key physiological roles in the central and peripheral nervous systems. The prototypical agonist nicotine acts intracellularly to upregulate many nAChR subtypes, a phenomenon that is thought to contribute to the nicotine dependence of cigarette smokers. The α3ß4 subtype has recently been genetically linked to nicotine dependence and lung cancer; however, the mode of action of nicotine on this receptor subtype has been incompletely investigated. Here, using transfected mammalian cells as model system, we characterized the response of the human α3ß4 receptor subtype to nicotine and the mechanism of action of the drug. Nicotine, when present at 1 mm concentration, elicited a â¼5-fold increase of cell surface α3ß4 and showed a more modest upregulatory effect also at concentrations as low as 10 µM. Upregulation was obtained if nicotine was present during, but not after, pentamer assembly and was caused by increased stability and trafficking of receptors assembled in the presence of the drug. Experimental determinations as well as computational studies of subunit stoichiometry showed that nicotine favors assembly of pentamers with (α3)2(ß4)3 stoichiometry; these are less prone than (α3)3(ß4)2 receptors to proteasomal degradation and, because of the presence in the ß subunit of an endoplasmic reticulum export motif, more efficiently transported to the plasma membrane. Our findings uncover a novel mechanism of nicotine-induced α3ß4 nAChR upregulation that may be relevant also for other nAChR subtypes.
Asunto(s)
Nicotina/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptores Nicotínicos/metabolismo , Fumar/fisiopatología , Animales , Anticuerpos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Masculino , Modelos Químicos , Mutagénesis/fisiología , Neuroblastoma , Agonistas Nicotínicos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Conejos , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/inmunología , Fumar/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
VAPB (vesicle-associated membrane protein-associated protein B) is a ubiquitously expressed, ER-resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. Its mutant form, P56S-VAPB, is linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently demonstrated that these inclusions are formed by profoundly remodelled stacked ER cisternae. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild-type VAPB and P56S-VAPB, as well as microinjection protocols in non-transfected cells, to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then congregate in the juxtanuclear region independently of the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wild-type protein, so that the inclusions are cleared only a few hours after cessation of P56S-VAPB synthesis. At variance with other inclusion bodies linked to neurodegenerative diseases, clearance of P56S-VAPB inclusions involves the proteasome, with no apparent participation of macro-autophagy. Transfection of a dominant-negative form of the AAA ATPase p97/VCP stabilizes mutant VAPB, suggesting a role for this ATPase in extracting the aggregated protein from the inclusions. Our results demonstrate that the structures induced by P56S-VAPB stand apart from other inclusion bodies, both in the mechanism of their genesis and of their clearance from the cell, with possible implications for the pathogenic mechanism of the mutant protein.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Retículo Endoplásmico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Línea Celular , Retículo Endoplásmico/genética , Células HeLa , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Transporte de Proteínas , Transfección , Ubiquitinación , Proteínas de Transporte Vesicular/genéticaRESUMEN
A large group of diverse, functionally important, and differently localized transmembrane proteins shares a particular membrane topology, consisting of a cytosolic N-terminal region, followed by a transmembrane domain close to the C-terminus. Because of their structure, these C-tail-anchored (TA) proteins must insert into all their target membranes by post-translational pathways. Recent work, based on the development of stringent and sensitive biochemical assays, has demonstrated that novel unexplored mechanisms underlie these post-translational targeting and membrane insertion pathways. Unravelling these pathways will shed light on the biosynthesis and regulation of an important group of membrane proteins and is likely to lead to new concepts in the field of membrane biogenesis.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Modelos Biológicos , Animales , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína , Transporte de Proteínas , Transducción de SeñalRESUMEN
It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an N-terminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5)tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three- to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFP-b(5)tail expression; peak ATF6α activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6α abolished the ER proliferation response, whereas knockdown of Ire1 was without effect. Because GFP-b(5)tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Bases , Calcio/metabolismo , Citocromos b5/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Doxiciclina/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Proteínas Fluorescentes Verdes/fisiología , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Modelos Biológicos , Fosfatidilcolinas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismoRESUMEN
Chemical chaperones are small molecules that improve protein folding, alleviating aberrant pathological phenotypes due to protein misfolding. Recent reports suggest that, in parallel with their role in relieving endoplasmic reticulum (ER) stress, chemical chaperones rescue mitochondrial function and insulin signaling. These effects may underlie their pharmacological action on metabolically demanding tissues.
Asunto(s)
Chaperonas Moleculares , Pliegue de Proteína , Humanos , Animales , Chaperonas Moleculares/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Mitocondrias/metabolismo , Insulina/metabolismoRESUMEN
Genome-wide association studies have led to the identification of numerous susceptibility genes for type 2 diabetes. Among them is Cdkal1, which is associated with reduced ß-cell function and insulin release. Recently, CDKAL1 has been shown to be a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Here, we report that out of several CDKAL1 isoforms deposited in public databases, only isoform 1, which migrates as a 61-kDa protein by SDS-PAGE, is expressed in human islets and pancreatic insulinoma INS-1 and MIN6 cells. We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. Using endo-ß-N-acetylglucosaminidase H and peptide:N-glycosidase F sensitivity assays on CDKAL1 constructs carrying an N-glycosylation site within the luminal domain, we further established that CDKAL1 is an endoplasmic reticulum-resident protein. Moreover, we observed that silencing CDKAL1 in INS-1 cells reduces the expression of secretory granule proteins prochromogranin A and proICA512/ICA512-TMF, in addition to proinsulin and insulin. This correlated with reduced glucose-stimulated insulin secretion. Taken together, our findings provide new insight into the role of CDKAL1 in insulin-producing cells and help to understand its involvement in the pathogenesis of diabetes.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Retículo Endoplásmico/metabolismo , Insulinoma/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Quinasa 5 Dependiente de la Ciclina/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Silenciador del Gen , Humanos , Insulinoma/genética , Insulinoma/patología , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proinsulina/genética , Proinsulina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Ratas , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/metabolismo , ARNt MetiltransferasasRESUMEN
The endoplasmic reticulum (ER) is a dynamic pleiomorphic organelle containing continuous but distinct subdomains. The diversity of ER structures parallels its many functions, including secretory protein biogenesis, lipid synthesis, drug metabolism and Ca2+ signaling. Recent studies are revealing how elaborate ER structures arise in response to subtle changes in protein levels, dynamics, and interactions as well as in response to alterations in cytosolic ion concentrations. Subdomain formation appears to be governed by principles of self-organization. Once formed, ER subdomains remain malleable and can be rapidly transformed into alternative structures in response to altered conditions. The mechanisms that modulate ER structure are likely to be important for the generation of the characteristic shapes of other organelles.
Asunto(s)
Retículo Endoplásmico/ultraestructura , Animales , Retículo Endoplásmico/fisiología , HumanosRESUMEN
Sensorineural age-related hearing loss affects a large proportion of the elderly population, and has both environmental and genetic causes. Notwithstanding increasing interest in this debilitating condition, the genetic risk factors remain largely unknown. Here, we report the case of two sisters affected by isolated profound sensorineural hearing loss after the age of seventy. Genomic DNA sequencing revealed that the siblings shared two monoallelic variants in two genes linked to Usher Syndrome (USH genes), a recessive disorder of the ear and the retina: a rare pathogenic truncating variant in USH1G and a previously unreported missense variant in ADGRV1. Structure predictions suggest a negative effect on protein stability of the latter variant, allowing its classification as likely pathogenic according to American College of Medical Genetics criteria. Thus, the presence in heterozygosis of two recessive alleles, which each cause syndromic deafness, may underlie digenic inheritance of the age-related non-syndromic hearing loss of the siblings, a hypothesis that is strengthened by the knowledge that the two genes are integrated in the same functional network, which underlies stereocilium development and organization. These results enlarge the spectrum and complexity of the phenotypic consequences of USH gene mutations beyond the simple Mendelian inheritance of classical Usher syndrome.
RESUMEN
C-tail-anchored (TA) proteins constitute a heterogeneous group of membrane proteins that are inserted into membranes by unique post-translational mechanisms and that play key roles within cells. During recent years, bioinformatic screens on eukaryotic genomes have helped to obtain comprehensive pictures of the number, intracellular distribution and functions of TA proteins, but similar screens had not yet been carried out on prokaryotic cells. Here, we report the results of a bioinformatic screen of the genomes of two bacteria and one archeon. We find that all three of these prokaryotes contain TA proteins in proportions approaching those found in eukaryotic cells, indicating that this protein group is present in all three domains of life. Although some of our hits correspond to proteins of unknown function, others are enzymes with hydrophobic substrates or have functions carried out at the inner face of the cytoplasmic membrane. To generate hypotheses on the insertion mechanisms of prokaryotic TA proteins, we compared the sequences of the prokaryotic and eukaryotic versions of Asna1/Trc40/GET3, a cytosolic ATPase that plays a key role in TA protein post-translational delivery to membranes in eukaryotic cells. We found that hydrophobic residues involved in TA binding by the eukaryotic chaperone (Mateja et al., Nature 2009;461:361-366) are generally replaced with equally hydrophobic amino acids in the archeal homologue (ArsA), whereas this is not the case for the bacterial protein. Thus, eukaryotes may have inherited the GET3 targeting pathway from our archeal ancestor, while the bacterial homologue may be exclusively dedicated to heavy metal resistance.