RESUMEN
Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through the activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of overexpression of SARS-CoV-2 nsp4, a key driver of replication, on the UPR in cell culture using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find that nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found that an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their coexpression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Reanalysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Proteómica , Respuesta de Proteína Desplegada , Técnicas de Cultivo de CélulaRESUMEN
The erlin1/erlin2 (E1/E2) complex is an endoplasmic reticulum membrane-located assemblage of the proteins erlin1 and erlin2. Here, we demonstrate direct and selective binding of phosphatidylinositol 3-phosphate (PI(3)P) to recombinant erlins and that disruption or deletion of the E1/E2 complex reduces HeLa cell PI(3)P levels by â¼50 %. This reduction correlated with a decrease in autophagic flux, with no effect on the endocytic pathway, and was not due to reduced VPS34 kinase activity, which is critical for maintaining steady-state PI(3)P levels. Pharmacological inhibition of VPS34 and suppression of PI(3)P levels caused a similar reduction in autophagic flux. Overall, these data indicate that by binding to PI(3)P, the E1/E2 complex plays an important role in maintaining the steady-state levels of PI(3)P and, thus, sustains some key PI(3)P-dependent processes, e.g., autophagy.
Asunto(s)
Autofagia , Fosfatos de Fosfatidilinositol , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Células HeLa , Proteínas de la Membrana/metabolismo , Unión Proteica , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Retículo Endoplásmico/metabolismoRESUMEN
Long-term activation of inositol 1,4,5-trisphosphate receptors (IP3Rs) leads to their degradation by the ubiquitin-proteasome pathway. The first and rate-limiting step in this process is thought to be the association of conformationally active IP3Rs with the erlin1/2 complex, an endoplasmic reticulum-located oligomer of erlin1 and erlin2 that recruits the E3 ubiquitin ligase RNF170, but the molecular determinants of this interaction remain unknown. Here, through mutation of IP3R1, we show that the erlin1/2 complex interacts with the IP3R1 intralumenal loop 3 (IL3), the loop between transmembrane (TM) helices 5 and 6, and in particular, with a region close to TM5, since mutation of amino acids D-2471 and R-2472 can specifically block erlin1/2 complex association. Surprisingly, we found that additional mutations in IL3 immediately adjacent to TM5 (e.g., D2465N) almost completely abolish IP3R1 Ca2+ channel activity, indicating that the integrity of this region is critical to IP3R1 function. Finally, we demonstrate that inhibition of the ubiquitin-activating enzyme UBE1 by the small-molecule inhibitor TAK-243 completely blocked IP3R1 ubiquitination and degradation without altering erlin1/2 complex association, confirming that association of the erlin1/2 complex is the primary event that initiates IP3R1 processing and that IP3R1 ubiquitination mediates IP3R1 degradation. Overall, these data localize the erlin1/2 complex-binding site on IP3R1 to IL3 and show that the region immediately adjacent to TM5 is key to the events that facilitate channel opening.
Asunto(s)
Retículo Endoplásmico , Receptores de Inositol 1,4,5-Trifosfato , Proteínas de la Membrana , Ubiquitina , Animales , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Bcl-2-related ovarian killer (Bok) binds tightly to inositol 1,4,5-trisphosphate receptors (IP3Rs). To better understand this interaction, we sought to elucidate the Bok binding determinants in IP3R1, focusing on the â¼75 amino acid loop (residues 1882-1957) between α helices 72 and 73. Bioinformatic analysis revealed that the majority of this loop is intrinsically disordered, with two flanking regions of high disorder next to a low disorder central region (â¼residues 1914-1926) that is predicted to contain two fused, disjointed transient helical elements. Experiments with IP3R1 mutants, combined with computational analysis, indicated that small deletions in this central region block Bok binding due to perturbation of the helical elements. Studies in vitro with purified Bok and IP3R1-derived peptides revealed high affinity binding to amino acids 1898-1940 of IP3R1 (Kd â¼65 nM) and that binding affinity is also dependent upon both of the high disorder flanking regions. The strength of the Bok-IP3R1 interaction was demonstrated by the ability of IP3R1 or Bok to recruit transmembrane domain-free Bok or IP3R1 mutants, respectively, to membranes in intact cells, and that these two mutants can bind in the cytosol independently of membrane association. Overall, we show that Bok binding to IP3R1 occurs within a largely disordered loop between α helices 72 and 73 and that high affinity binding is mediated by multivalent interactions.
Asunto(s)
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sitios de Unión/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Modelos Moleculares , Terapia Molecular Dirigida , Unión Proteica/genética , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/química , Eliminación de SecuenciaRESUMEN
The erlin1/2 complex is a â¼2-MDa endoplasmic reticulum membrane-located ensemble of the â¼40-kDa type II membrane proteins erlin1 and erlin2. The best defined function of this complex is to mediate the ubiquitination of activated inositol 1,4,5-trisphosphate receptors (IP3Rs) and their subsequent degradation. However, it remains unclear how mutations of the erlin1/2 complex affect its cellular function and cause cellular dysfunction and diseases such as hereditary spastic paraplegia. Here, we used gene editing to ablate erlin1 or erlin2 expression to better define their individual roles in the cell and examined the functional effects of a spastic paraplegia-linked mutation to erlin2 (threonine to isoleucine at position 65; T65I). Our results revealed that erlin2 is the dominant player in mediating the interaction between the erlin1/2 complex and IP3Rs and that the T65I mutation dramatically inhibits this interaction and the ability of the erlin1/2 complex to promote IP3R ubiquitination and degradation. Remarkably, we also discovered that the erlin1/2 complex specifically binds to phosphatidylinositol 3-phosphate, that erlin2 binds this phospholipid much more strongly than does erlin1, that the binding is inhibited by T65I mutation of erlin2, and that multiple determinants within the erlin2 polypeptide comprise the phosphatidylinositol 3-phosphate-binding site. Overall, these results indicate that erlin2 is the primary mediator of the cellular roles of the erlin1/2 complex and that disease-linked mutations of erlin2 can affect both IP3R processing and lipid binding.
Asunto(s)
Sustitución de Aminoácidos , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Fosfatos de Fosfatidilinositol/metabolismo , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas , Línea Celular Transformada , Edición Génica , Gonadotrofos/citología , Gonadotrofos/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de la Membrana/deficiencia , Ratones , Mutación , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Proteolisis , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismo , Paraplejía Espástica Hereditaria/patología , UbiquitinaciónRESUMEN
Bok is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, although the role that Bok plays in this pathway is unclear. We have shown previously in cultured cell lines that Bok interacts strongly with inositol 1,4,5-trisphosphate receptors (IP3Rs), suggesting that it may contribute to the structural integrity or stability of IP3R tetramers. Here we report that Bok is similarly IP3R-assocated in mouse tissues, that essentially all cellular Bok is IP3R bound, that it is the helical nature of the Bok BH4 domain, rather than specific amino acids, that mediates binding to IP3Rs, that Bok is dramatically stabilized by binding to IP3Rs, that unbound Bok is ubiquitinated and degraded by the proteasome, and that binding to IP3Rs limits the pro-apoptotic effect of overexpressed Bok. Agents that stimulate IP3R activity, apoptosis, phosphorylation, and endoplasmic reticulum stress did not trigger the dissociation of mature Bok from IP3Rs or Bok degradation, indicating that the role of proteasome-mediated Bok degradation is to destroy newly synthesized Bok that is not IP3R associated. The existence of this unexpected proteolytic mechanism that is geared toward restricting Bok to that which is bound to IP3Rs, implies that unbound Bok is deleterious to cell viability and helps explain the current uncertainty regarding the cellular role of Bok.
Asunto(s)
Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Señalización del Calcio , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Inmunoprecipitación , Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Noqueados , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación/genética , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca(2+) mobilization was significantly impaired, whereas neither Ca(2+) store content, IP3 receptor levels, nor IP3 production were altered, indicative of a functional defect at the IP3 receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP3 receptors (monoubiquitin and Lys(48)- and Lys(63)-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP3 receptors. Thus, the Ca(2+) mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP3 receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels.
Asunto(s)
Ataxia/congénito , Señalización del Calcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mutación Puntual , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Arginina/química , Ataxia/genética , Ataxia/metabolismo , Calcio/química , Línea Celular , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Exones , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Lisina/química , Ratones , Mutación , Enfermedades Neurodegenerativas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895-1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.
Asunto(s)
Apoptosis/fisiología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Calcio/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Transformada , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones , Ratones Noqueados , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Bcl-2-related ovarian killer, Bok, was first labeled "pro-apoptotic" due to its ability to cause cell death when over-expressed. However, it has become apparent that this is not a good name, since Bok is widely expressed in tissues other than ovaries. Further, there is serious doubt as to whether Bok is a real "killer," due to disparities in the ability of over-expressed versus endogenous Bok to trigger apoptosis. In this brief review, we rationalize these disparities and argue that endogenous Bok is very different from the pro-apoptotic, mitochondrial outer membrane permeabilization mediators, Bak and Bax. Instead, Bok is a stable, endoplasmic reticulum-located protein bound to inositol 1,4,5 trisphosphate receptors. From this location, Bok plays a variety of roles, including regulation of endoplasmic reticulum/mitochondria contact sites and mitochondrial dynamics. Therefore, categorizing Bok as a "killer" may well be misleading and instead, endogenous Bok would better be considered an endoplasmic reticulum-located "bystander", with non-apoptotic roles.
RESUMEN
Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of SARS-CoV-2 nsp4, a key driver of replication, on the UPR using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their co-expression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Re-analysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.
RESUMEN
Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP(3) and Ca(2+)-gated Ca(2+) channels. Endogenous IP(3)Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP(3)R1 in αT3-1 pituitary gonadotrophs and found that IP(3)R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D. A., Kubota, K., Kirkpatrick, D. S., Alzayady, K. J., Gygi, S. P., and Wojcikiewicz, R. J. H. (2008) J. Biol. Chem. 283, 35319-35328). Here, we have extended these studies to determine whether IP(3)R2 and IP(3)R3 are similarly modified and if ubiquitination is cell type-dependent. Using mass spectrometry and linkage-specific ubiquitin antibodies, we found that all IP(3)R types are subject to ubiquitination at approximately the same locations and that, independent of cell type, IP(3)Rs are modified by monoubiquitin and Lys-48- and Lys-63-linked ubiquitin chains, although in differing proportions. Remarkably, the attached Lys-48- and Lys-63-linked ubiquitin chains are homogeneous and are segregated to separate IP(3)R subunits, and Lys-48-linked ubiquitin chains, but not Lys-63-linked chains, are required for IP(3)R degradation. Together, these data provide unique insight into the complexities of ubiquitination of an endogenous ubiquitin-proteasome pathway substrate in unperturbed mammalian cells. Importantly, although Lys-48-linked ubiquitin chains appear to trigger proteasomal degradation, the presence of Lys-63-linked ubiquitin chains suggests that ubiquitination of IP(3)Rs may have physiological consequences beyond signaling for degradation.
Asunto(s)
Canales de Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Línea Celular Tumoral , Fibroblastos/citología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Lisina/metabolismo , Ratones , Datos de Secuencia Molecular , Neuroblastoma , Neoplasias Pancreáticas , Hipófisis/citología , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , Ratas , Receptores Citoplasmáticos y Nucleares/química , UbiquitinaciónRESUMEN
Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum membrane calcium channels that, upon activation, are degraded via the ubiquitin-proteasome pathway. While searching for novel mediators of IP(3) receptor processing, we discovered that RNF170, an uncharacterized RING domain-containing protein, associates rapidly with activated IP(3) receptors. RNF170 is predicted to have three membrane-spanning helices, is localized to the ER membrane, and possesses ubiquitin ligase activity. Depletion of endogenous RNF170 by RNA interference inhibited stimulus-induced IP(3) receptor ubiquitination, and degradation and overexpression of a catalytically inactive RNF170 mutant suppressed stimulus-induced IP(3) receptor processing. A substantial proportion of RNF170 is constitutively associated with the erlin1/2 (SPFH1/2) complex, which has been shown previously to bind to IP(3) receptors immediately after their activation. Depletion of RNF170 did not affect the binding of the erlin1/2 complex to stimulated IP(3) receptors, whereas erlin1/2 complex depletion inhibited RNF170 binding. These results suggest a model in which the erlin1/2 complex recruits RNF170 to activated IP(3) receptors where it mediates IP(3) receptor ubiquitination. Thus, RNF170 plays an essential role in IP(3) receptor processing via the ubiquitin-proteasome pathway.
Asunto(s)
Retículo Endoplásmico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Animales , Retículo Endoplásmico/genética , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Controversy surrounds the cellular role of the Bcl-2 family protein Bok. On one hand, it has been shown that all endogenous Bok is bound to inositol 1,4,5-trisphosphate receptors (IP3Rs), while other data suggest that Bok can act as a pro-apoptotic mitochondrial outer membrane permeabilization mediator, apparently kept at very low and non-apoptotic levels by efficient proteasome-mediated degradation. Here we show that 1) endogenous Bok is expressed at readily-detectable levels in key cultured cells (e.g., mouse embryonic fibroblasts and HCT116 cells) and is not constitutively degraded by the proteasome, 2) proteasome inhibitor-induced apoptosis is not mediated by Bok, 3) endogenous Bok expression level is critically dependent on the presence of IP3Rs, 4) endogenous Bok is rapidly degraded by the ubiquitin-proteasome pathway in the absence of IP3Rs at the endoplasmic reticulum membrane, and 5) charged residues in the transmembrane region of Bok affect its stability, ability to interact with Mcl-1, and pro-apoptotic activity when over-expressed. Overall, these data indicate that endogenous Bok levels are not governed by proteasomal activity (except when IP3Rs are deleted) and that while endogenous Bok plays little or no role in apoptotic signaling, exogenous Bok can mediate apoptosis in a manner dependent on its transmembrane domain.
RESUMEN
Erlin1 and erlin2 are highly homologous, â¼40kDa, endoplasmic reticulum membrane proteins that assemble into a ring-shaped complex with a mass of â¼2 MDa. How this complex is formed is not understood, but appears to involve multiple interactions, including a coiled-coil region that mediates lower-order erlin assembly, and a short hydrophobic region, termed the "assembly domain", that mediates higher-order assembly into â¼2 MDa complexes. Here we have used molecular modeling, mutagenesis and cross-linking to examine the role of the assembly domain in higher-order assembly. We find (i) that the assembly domains of erlin1 and erlin2 are amphipathic helices, (ii) that erlin1 alone and erlin2 alone can assemble into â¼2 MDa complexes, (iii) that higher-order assembly is strongly inhibited by point mutations to the assembly domain, (iv) that three interacting hydrophobic residues in the assembly domain and aromaticity are essential for higher-order assembly, and (iv) that while erlins1 and 2 are equally capable of forming lower-order homo- and hetero-oligomers, hetero-oligomers are the most prevalent form when erlin1 and erlin2 are co-expressed. Overall, we conclude that the â¼2 MDa erlin1/2 complex is composed of an assemblage of lower-order hetero-oligomers, probably heterotrimers, linked together by assembly domain hydrophobic residues.
Asunto(s)
Aminoácidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Secuencia de Aminoácidos , Aminoácidos/genética , Animales , Células HeLa , Humanos , Proteínas de la Membrana/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
The function of the Bcl-2 family member Bok is currently enigmatic, with various disparate roles reported, including mediation of apoptosis, regulation of mitochondrial morphology, binding to inositol 1,4,5-trisphosphate receptors, and regulation of uridine metabolism. To better define the roles of Bok, we examined its interactome using TurboID-mediated proximity labeling in HeLa cells, in which Bok knock-out leads to mitochondrial fragmentation and Bok overexpression leads to apoptosis. Labeling with TurboID-Bok revealed that Bok was proximal to a wide array of proteins, particularly those involved in mitochondrial fission (e.g., Drp1), endoplasmic reticulum-plasma membrane junctions (e.g., Stim1), and surprisingly among the Bcl-2 family members, just Mcl-1. Comparison with TurboID-Mcl-1 and TurboID-Bak revealed that the three Bcl-2 family member interactomes were largely independent, but with some overlap that likely identifies key interactors. Interestingly, when overexpressed, Mcl-1 and Bok interact physically and functionally, in a manner that depends upon the transmembrane domain of Bok. Overall, this work shows that the Bok interactome is different from those of Mcl-1 and Bak, identifies novel proximities and potential interaction points for Bcl-2 family members, and suggests that Bok may regulate mitochondrial fission via Mcl-1 and Drp1.
RESUMEN
Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. While it is clear that IP(3) receptors are polyubiquitinated and are transferred to the proteasome by a p97-based complex, currently very little is known about the proteins that initially select activated IP(3) receptors for ERAD. Here, we have transfected HeLa cells to stably express m3 muscarinic receptors to allow for the study of IP(3) receptor ERAD in this cell type, and show that IP(3) receptors are polyubiquitinated and then degraded by the proteasome in response to carbachol, a muscarinic agonist. In seeking to identify proteins that mediate IP(3) receptor ERAD we found that both SPFH1 and SPFH2 (also known as erlin 1 and erlin 2), which exist as a hetero-oligomeric complex, rapidly associate with IP(3) receptors in a manner that precedes polyubiquitination and the association of p97. Suppression of SPFH1 and SPFH2 expression by RNA interference markedly inhibited carbachol-induced IP(3) receptor polyubiquitination and degradation, but did not affect carbachol-induced calcium mobilization or IkappaBalpha processing, indicating that the SPFH1/2 complex is a key player in IP(3) receptor ERAD, acting at a step after IP(3) receptor activation, but prior to IP(3) receptor polyubiquitination. Suppression of SPFH1 and SPFH2 expression had only slight effects on the turnover of some exogenous model ERAD substrates, and had no effect on sterol-induced ERAD of endogenous 3-hydroxy-3-methylglutaryl-CoA reductase. Overall, these studies show that m3 receptor-expressing HeLa cells are a valuable system for studying IP(3) receptor ERAD, and suggest that the SPFH1/2 complex is a factor that selectively mediates the ERAD of activated IP(3) receptors.
Asunto(s)
Expresión Génica , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Muscarínico M3/biosíntesis , Ubiquitinación/fisiología , Células HeLa , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso , Receptor Muscarínico M3/genéticaRESUMEN
IP3 receptor turnover is mediated by the ubiquitin ligase RNF170, which is recruited to active IP3 receptors by the erlin1/2 complex. A new study by Wagner et al (Nat Commun, 2019) links four cases of Hereditary Spastic Paraplegia to inactivating mutations in RNF170. This increases the number of examples of mutations to the erlin1/2 complex-RNF170 module underlying neurodegenerative disorders.
Asunto(s)
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Paraplejía Espástica Hereditaria/metabolismo , Humanos , Modelos Biológicos , Mutación , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The epsin family of endocytic adapter proteins are widely expressed, and interact with both proteins and lipids to regulate a variety of cell functions. However, the role of epsins in atherosclerosis is poorly understood. Here, we show that deletion of endothelial epsin proteins reduces inflammation and attenuates atherosclerosis using both cell culture and mouse models of this disease. In atherogenic cholesterol-treated murine aortic endothelial cells, epsins interact with the ubiquitinated endoplasmic reticulum protein inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which triggers proteasomal degradation of this calcium release channel. Epsins potentiate its degradation via this interaction. Genetic reduction of endothelial IP3R1 accelerates atherosclerosis, whereas deletion of endothelial epsins stabilizes IP3R1 and mitigates inflammation. Reduction of IP3R1 in epsin-deficient mice restores atherosclerotic progression. Taken together, epsin-mediated degradation of IP3R1 represents a previously undiscovered biological role for epsin proteins and may provide new therapeutic targets for the treatment of atherosclerosis and other diseases.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Aterosclerosis/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteolisis , Proteínas Adaptadoras del Transporte Vesicular/química , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/patología , Calcio/metabolismo , Colesterol/metabolismo , Células Endoteliales/metabolismo , Femenino , Eliminación de Gen , Células HEK293 , Homeostasis , Humanos , Inflamación/patología , Masculino , Ratones Noqueados , Unión Proteica , Dominios Proteicos , UbiquitinaciónRESUMEN
Reduction in endothelium-derived hyperpolarizing factor (EDHF)-mediated dilatory function in large, elastic arteries during hypertension is reversed after blood pressure normalization. We investigated whether similar mechanisms occurred in smaller mesenteric resistance arteries from aged Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs), and SHRs treated with the angiotensin-converting enzyme inhibitor, enalapril, using immunohistochemistry, serial-section electron microscopy, electrophysiology and wire myography. Unlike the superior mesenteric artery, EDHF relaxations in muscular mesenteric arteries were not reduced in SHRs, although morphological differences were found in the endothelium and smooth muscle. In WKY rats, SHRs and enalapril-treated SHRs, relaxations were mediated by small-, large-, and intermediate-conductance calcium-activated potassium channels, which were distributed in the endothelium, smooth muscle, and both layers, respectively. However, only WKY hyperpolarizations and relaxations were sensitive to gap junction blockers, and these arteries expressed more endothelial and myoendothelial gap junctions than arteries from SHRs. Responses in WKY rats, but not SHRs, were also reduced by inhibitors of epoxyeicosatrienoic acids (EETs), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) and miconazole, although sensitivity to EET regioisomers was endothelium-independent in all rats. Enalapril treatment of SHRs reduced blood pressure and restored sensitivity to 14,15-EEZE, but not to gap junction blockers, and failed to reverse the morphological changes. In conclusion, the mechanisms underlying EDHF in muscular mesenteric arteries differ between WKY rats and SHRs, with gap junctions and EETs involved only in WKY rats. However, reduction of blood pressure in SHRs with enalapril restored a role for EETs, but not gap junctions, without reversing morphological changes, suggesting a differential control of chemical and structural alterations.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Factores Biológicos/metabolismo , Enalapril/uso terapéutico , Uniones Comunicantes/metabolismo , Hipertensión/metabolismo , Arterias Mesentéricas/metabolismo , Animales , Enalapril/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/ultraestructura , Hipertensión/tratamiento farmacológico , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/ultraestructura , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
Bok (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, but the cellular role that Bok plays is controversial. Remarkably, endogenous Bok is constitutively bound to inositol 1,4,5-trisphosphate receptors (IP3Rs) and is stabilized by this interaction. Here we report that despite the strong association with IP3Rs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IP3Rs or calcium influx into the mitochondria. Rather, Bok deletion significantly reduces mitochondrial fusion rate, resulting in mitochondrial fragmentation. This phenotype is reversed by exogenous wild-type Bok and by an IP3R binding-deficient Bok mutant, and may result from a decrease in mitochondrial motility. Bok deletion also enhances mitochondrial spare respiratory capacity and membrane potential. Finally, Bok does not play a major role in apoptotic signaling, since Bok deletion does not alter responsiveness to various apoptotic stimuli. Overall, despite binding to IP3Rs, Bok does not alter IP3R-mediated Ca2+ signaling, but is required to maintain normal mitochondrial fusion, morphology, and bioenergetics.