RESUMEN
Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.
Asunto(s)
Proteína Fosfatasa 2 , Proteína Fosfatasa 2/metabolismo , Jordania , Fosforilación , Mutación , Holoenzimas/genética , Holoenzimas/metabolismoRESUMEN
Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Anticuerpos de Dominio Único , Animales , Ratones , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Enterotoxinas/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Epítopos/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
IMPORTANCE: The COVID-19 pandemic exposed limitations of conventional antibodies as therapeutics, including high cost, limited potency, ineffectiveness against new viral variants, and primary reliance on injection-only delivery. Nanobodies are single-domain antibodies with therapeutic potentials. We discovered three anti-SARS-CoV-2 nanobodies, named Nanosota-2, -3, and -4, from an immunized alpaca. Nanosota-2 is super potent against prototypic SARS-CoV-2, Nanosota-3 is highly potent against the omicron variant, and Nanosota-4 is effective against both SARS-CoV-1 and SARS-CoV-2. In addition to their super potency and combined broad antiviral spectrum, these nanobodies are cost-effective, can be easily adapted to new viral variants through phage display, and can potentially be administered as inhalers. The Nanosota series are powerful therapeutic candidates to combat circulating SARS-CoV-2 and prepare for possible future coronavirus pandemics.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos de Dominio Único , Humanos , Anticuerpos Neutralizantes , Anticuerpos Antivirales/uso terapéutico , COVID-19/terapia , Pandemias , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del CoronavirusRESUMEN
Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer's disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.
Asunto(s)
Proteína Fosfatasa 2 , Proteínas Proto-Oncogénicas c-fyn , Familia-src Quinasas , Enfermedad de Alzheimer/metabolismo , Humanos , Fosfoproteínas Fosfatasas , Fosforilación , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo , Proteínas tau/metabolismoRESUMEN
Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACß > PP2ACα > PP4C > PP6C), NRVM (PP2ACß > PP2ACα = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACα > PP2ACß > PP6C > PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACα, PP2ACß, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (γH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of γH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium.
Asunto(s)
Hipertrofia Ventricular Izquierda/enzimología , Miocitos Cardíacos/enzimología , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Animales Recién Nacidos , Línea Celular , Daño del ADN , Regulación Enzimológica de la Expresión Génica , Histonas/metabolismo , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Chaperonas Moleculares , Miocitos Cardíacos/patología , Estrés Oxidativo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Fosfatasa 2/genética , Interferencia de ARN , Ratas Sprague-Dawley , Ratas Wistar , TransfecciónRESUMEN
Alpha4 is a non-canonical regulatory subunit of Type 2A protein phosphatases that interacts directly with the phosphatase catalytic subunits (PP2Ac, PP4c, and PP6c) and is upregulated in a variety of cancers. Alpha4 modulates phosphatase expression levels and activity, but the molecular mechanism of this regulation is unclear, and the extent to which the various Type 2A catalytic subunits associate with Alpha4 is also unknown. To determine the relative fractions of the Type 2A catalytic subunits associated with Alpha4, we conducted Alpha4 immunodepletion experiments in HEK293T cells and found that a significant fraction of total PP6c is associated with Alpha4, whereas a minimal fraction of total PP2Ac is associated with Alpha4. To facilitate studies of phosphatases in the presence of mutant or null Alpha4 alleles, we developed a facile and rapid method to simultaneously knockdown and rescue Alpha4 in tissue culture cells. This approach has the advantage that levels of endogenous Alpha4 are dramatically reduced by shRNA expression thereby simplifying interpretation of mutant phenotypes. We used this system to show that knockdown of Alpha4 preferentially impacts the expression of PP4c and PP6c compared to expression levels of PP2Ac.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Dominio Catalítico , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/genética , Chaperonas Moleculares , Fosfoproteínas Fosfatasas/análisis , Proteína Fosfatasa 2/análisisRESUMEN
Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glicoproteínas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismo , Sustitución de Aminoácidos , Animales , Bovinos , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/genética , Glicoproteínas/genética , Humanos , Complejos Multienzimáticos/genética , Mutación Missense , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Ratas , Proteína de Unión al GTP rac1/genética , Proteínas ras/genéticaRESUMEN
The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Placa Amiloide , Anticuerpos de Dominio Único , Animales , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Ratones , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Humanos , Encéfalo/metabolismo , Encéfalo/patología , Barrera Hematoencefálica/metabolismo , Ratones Transgénicos , Camélidos del Nuevo Mundo , Modelos Animales de EnfermedadRESUMEN
The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.
RESUMEN
Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Fosfatasa 2/metabolismo , Ubiquitinación/fisiología , Western Blotting , Línea Celular , Humanos , Inmunoprecipitación , Espectrometría de Masas , Proteínas Asociadas a Microtúbulos/genética , Fosforilación/genética , Fosforilación/fisiología , Proteína Fosfatasa 2/genética , Estabilidad Proteica , Ubiquitinación/genéticaRESUMEN
Protein phosphorylation and dephosphorylation are both important for multiple steps in the splicing pathway. Members of the PP1 and PP2A subfamilies of phospho-serine/threonine phosphatases play essential but redundant roles in the second step of the splicing reaction. PP6, a member of the PP2A subfamily, is the mammalian homolog of yeast Sit4p and ppe1, which are involved in cell cycle regulation; however, the involvement of PP6 in the splicing pathway remains unclear. Here we show that PP2A family members physically associate with the spliceosome throughout the splicing reaction. PP2A holoenzyme and PP6 were found stably associated with U1 snRNP. Together our findings indicate that these phosphatases regulate splicing catalysis involving U1 snRNP and suggest an important evolutionary conserved role of PP2A family phosphatases in pre-mRNA splicing.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2/metabolismo , Empalme del ARN/fisiología , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Empalmosomas/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilación , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Timocitos/metabolismoRESUMEN
An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56δ (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56δ holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56δ termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.
RESUMEN
Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: 1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and 2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Chaperonas Moleculares , Fosfoproteínas/química , Fosfoproteínas/genética , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/genética , Estructura Terciaria de Proteína , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-D-ribose 5-phosphate (ribose 5-phosphate) and α-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator α-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn(2+)-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[(18)O(3)]phosphate and [U-(13)C(5)]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.
Asunto(s)
Fosfatasa Alcalina , Bacillus cereus/enzimología , Proteínas Bacterianas/química , Glucosa-6-Fosfato/análogos & derivados , Fosfotransferasas/química , Ribosamonofosfatos/química , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Glucosa-6-Fosfato/química , Glucosa-6-Fosfato/metabolismo , Manganeso/química , Manganeso/metabolismo , Fosfotransferasas/metabolismo , Ribosamonofosfatos/metabolismoRESUMEN
The current study examined the roles of Alpha4, a non-canonical subunit of protein phosphatase 2A, in the regulation of acute (insulin secretion) and chronic (cell dysfunction) effects of glucose in pancreatic beta cells. Alpha4 is expressed in human islets, rat islets and INS-1832/13 cells. Incubation of INS-1832/13 cells and rat islets with high glucose (HG) significantly increased the expression of Alpha4. C2-Ceramide, a biologically active sphingolipid, also increased the expression of Alpha4 in INS-1832/13 cells and rat islets. Subcellular distribution studies of Alpha4 in low glucose (LG) and HG exposed INS-1832/13 cells revealed that it is predominantly cytosolic, and its expression is significantly increased in the non-nuclear/cytosolic fractions in cells exposed to HG. siRNA-mediated knockdown of Alpha4 exerted minimal effects on glucose- or KCl-induced insulin secretion. siRNA-mediated deletion of Alpha4 significantly increased p38MAPK and JNK1/2 phosphorylation under LG conditions, comparable to the degree seen under HG conditions. Paradoxically, a significant potentiation of HG-induced p38MAPK and JNK2 phosphorylation was noted following Alpha4 deletion. HG-induced CHOP expression (ER stress marker) and caspase-3 activation were markedly attenuated in cells following Alpha4 knockdown. Deletion of Alpha4 in INS-1832/13 cells prevented HG-induced loss in the expression of Connexin36, a gap junction channel protein, which has been implicated in normal beta cell function. Lastly, depletion of endogenous Alpha4 significantly reduced HG-induced cell death in INS-1832/13 cells. Based on these findings we conclude that Alpha4 contributes to HG-induced metabolic dysfunction of the islet beta cell.
Asunto(s)
Células Secretoras de Insulina , Islotes Pancreáticos , Animales , Caspasa 3/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteína Fosfatasa 2/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Esfingolípidos/metabolismo , Esfingolípidos/farmacología , Estrés Fisiológico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Background: Ergothioneine (ERGO) is a unique antioxidant and a rare amino acid available in fungi and various bacteria but not in higher plants or animals. Substantial research data indicate that ERGO is a physiological antioxidant cytoprotectant. Different from other antioxidants that need to breach the blood-brain barrier to enter the brain parenchyma, a specialized transporter called OCTN1 has been identified for transporting ERGO to the brain. Purpose: To assess whether consumption of ERGO can prevent the progress of Alzheimer's disease (AD) on young (4-month-old) 5XFAD mice. Methods and materials: Three cohorts of mice were tested in this study, including ERGO-treated 5XFAD, non-treated 5XFAD, and WT mice. After the therapy, the animals went through various behavioral experiments to assess cognition. Then, mice were scanned with PET imaging to evaluate the biomarkers associated with AD using [11C]PIB, [11C]ERGO, and [18F]FDG radioligands. At the end of imaging, the animals went through cardiac perfusion, and the brains were isolated for immunohistology. Results: Young (4-month-old) 5XFAD mice did not show a cognitive deficit, and thus, we observed modest improvement in the treated counterparts. In contrast, the response to therapy was clearly detected at the molecular level. Treating 5XFAD mice with ERGO resulted in reduced amyloid plaques, oxidative stress, and rescued glucose metabolism. Conclusions: Consumption of high amounts of ERGO benefits the brain. ERGO has the potential to prevent AD. This work also demonstrates the power of imaging technology to assess response during therapy.
RESUMEN
The tumor suppressor, death-associated protein kinase (DAPK), is a Ca(2+)/calmodulin-regulated Ser/Thr kinase with an important role in regulating cytoskeletal dynamics. Autophosphorylation within the calmodulin-binding domain at Ser-308 inhibits DAPK catalytic activity. Dephosphorylation of Ser-308 by a previously unknown phosphatase enhances kinase activity and proteasome-mediated degradation of DAPK. In these studies, we identified two holoenzyme forms of protein phosphatase 2A (PP2A), ABalphaC and ABdeltaC, as DAPK-interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Ser-308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK levels by enhancing proteasome-mediated degradation of the kinase. Overexpression of wild type DAPK induces cell rounding and detachment in HEK293 cells; however, this effect is not observed following expression of an inactive DAPK S308E mutant. Finally, activation of DAPK by PP2A was found to be required for ceramide-induced anoikis. Together, our results provide a mechanism by which PP2A and DAPK activities control cell adhesion and anoikis.
Asunto(s)
Anoicis/efectos de los fármacos , Ceramidas/farmacología , Proteína Fosfatasa 2/metabolismo , Sustitución de Aminoácidos , Anoicis/genética , Proteínas Reguladoras de la Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular , Células HeLa , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Mutación Missense , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteína Fosfatasa 2/genética , Proteínas Supresoras de TumorRESUMEN
Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues 46-60) and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/metabolismo , Ubiquitinación/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Espectroscopía de Resonancia Magnética , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismo , Modelos Biológicos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitinación/genéticaRESUMEN
Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr307 but instead are hampered by PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr307 using targeted mass spectrometry after transient overexpression of PP2AC and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.
Asunto(s)
Anticuerpos/metabolismo , Especificidad de Anticuerpos/inmunología , Leucina/metabolismo , Fosfotirosina/metabolismo , Proteína Fosfatasa 2/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos/efectos de los fármacos , Reacciones Cruzadas/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Células HEK293 , Humanos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Metilación , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Péptidos/química , Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Vanadatos/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Drosophila visual signaling, a G-protein-coupled phospholipase Cbeta (PLCbeta)-mediated mechanism, is regulated by eye-protein kinase C (PKC) that promotes light adaptation and fast deactivation, most likely via phosphorylation of inactivation no afterpotential D (INAD) and TRP (transient receptor potential). To reveal the critical phosphatases that dephosphorylate INAD, we used several biochemical analyses and identified protein phosphatase 2A (PP2A) as a candidate. Importantly, the catalytic subunit of PP2A, microtubule star (MTS), is copurified with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in three mts heterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC [INAC (inactivation no afterpotential C] in vivo, we performed ERG recordings. We discovered that inaC(P209) was semidominant, because inaC(P209) heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of inaC(P209) heterozygotes was rescued by the mts(XE2258) heterozygous background. In contrast, mts(XE2258) failed to modify the severe deactivation of norpA(P16), indicating that MTS does not modulate NORPA (no receptor potential A) (PLCbeta). Together, our results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC, restoring the fast deactivation kinetics in vivo. We thus propose that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD.