RESUMEN
The post-translational modification of proteins with ubiquitin plays a central role in nearly all aspects of eukaryotic biology. Historically, studies have focused on the conjugation of ubiquitin to lysine residues in substrates, but it is now clear that ubiquitylation can also occur on cysteine, serine, and threonine residues, as well as on the N-terminal amino group of proteins. Paradigm-shifting reports of non-proteinaceous substrates have further extended the reach of ubiquitylation beyond the proteome to include intracellular lipids and sugars. Additionally, results from bacteria have revealed novel ways to ubiquitylate (and deubiquitylate) substrates without the need for any of the enzymatic components of the canonical ubiquitylation cascade. Focusing mainly upon recent findings, this review aims to outline the current understanding of non-lysine ubiquitylation and speculate upon the molecular mechanisms and physiological importance of this non-canonical modification.
RESUMEN
HOIL-1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL-1's E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL-1 monoubiquitylates glycogen and α1:4-linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester-linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100-fold by the interaction of Met1-linked or Lys63-linked ubiquitin oligomers with the RBR domain of HOIL-1. HOIL-1 also transferred pre-formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL-1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL-1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Glucanos , Glucosa , Glucógeno/metabolismo , Mamíferos , Ratones , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Components of bacteria and viruses activate Toll-Like Receptors in host cells, triggering the formation of the Myddosome and a signalling network that culminates in the production and release of the inflammatory mediators required to combat pathogenic infection. The Myddosome initiates signalling by recruiting and activating five E3 ligases that generate hybrid ubiquitin chains and attach them to components of the Myddosome. These ubiquitin chains act as a scaffold for the recruitment and activation of ubiquitin-binding proteins, which include the "master" protein kinases TAK1 and IKKß that drive inflammatory mediator production, as well as other proteins like ABIN1 and A20 that restrict activation of the network to prevent the overproduction of these substances that can lead to autoimmunity and organ damage. Here we review recent developments in our understanding of this network, focusing on the unexpected discovery that the E3 ligase HOIL-1 initiates the formation of hybrid ubiquitin chains by forming an ester bond between the first ubiquitin and the protein components of the Myddosome.
Asunto(s)
Factor 88 de Diferenciación Mieloide/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Factores de Transcripción/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1-generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism.
Asunto(s)
Inmunidad Innata , Complejos Multiproteicos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Catálisis , Técnicas de Sustitución del Gen , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Complejos Multiproteicos/fisiología , Serina , Treonina , UbiquitinaciónRESUMEN
Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about one-fifth of ubiquitin-dependent protein turnover in cells. CRLs are activated by the attachment of the ubiquitin-like protein neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to the cullin subunits. This cullin neddylation is essential for a plethora of CRL-regulated cellular processes and is vital for life. In mammals, neddylation is promoted by the five co-E3 ligases, defective in cullin neddylation 1 domain-containing 1-5 (DCNL1-5); however, their functional regulation within the CRL complex remains elusive. We found here that the ubiquitin-associated (UBA) domain-containing DCNL1 is monoubiquitylated when bound to CRLs and that this monoubiquitylation depends on the CRL-associated Ariadne RBR ligases TRIAD1 (ARIH2) and HHARI (ARIH1) and strictly requires the DCNL1's UBA domain. Reconstitution of DCNL1 monoubiquitylation in vitro revealed that autoubiquitylated TRIAD1 mediates binding to the UBA domain and subsequently promotes a single ubiquitin attachment to DCNL1 in a mechanism previously dubbed coupled monoubiquitylation. Moreover, we provide evidence that DCNL1 monoubiquitylation is required for efficient CRL activity, most likely by remodeling CRLs and their substrate receptors. Collectively, this work identifies DCNL1 as a critical target of Ariadne RBR ligases and coupled monoubiquitylation of DCNL1 as an integrated mechanism that affects CRL activity and client-substrate ubiquitylation at multiple levels.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteína NEDD8/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Portadoras/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína NEDD8/genética , Dominios Proteicos , Proteínas , Proteínas Proto-Oncogénicas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
N-terminal acetylation is an abundant modification influencing protein functions. Because â¼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Acetilación/efectos de los fármacos , Sitios de Unión , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Proteína NEDD8 , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.
Asunto(s)
Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Ligando RANK/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sustitución de Aminoácidos , Animales , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Noqueados , Mutación Missense , Factor 88 de Diferenciación Mieloide/genética , Proteínas Nucleares/genética , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ligando RANK/genética , Factor 6 Asociado a Receptor de TNF/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.
Asunto(s)
ADN/metabolismo , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/metabolismo , Línea Celular , Virus ADN/metabolismo , Expresión Génica , Humanos , Inmunidad Innata , Interferón beta/genética , Interferón beta/metabolismo , Queratinocitos/inmunología , Mutación , Proteínas Nucleares/genética , Nucleótidos Cíclicos/metabolismo , Fosfoproteínas/genética , Fosforilación , Transporte de ProteínasRESUMEN
Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation.
Asunto(s)
Proteínas Portadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Ubiquitinas/metabolismo , Proteínas Portadoras/genética , Proteínas Cullin/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mutación , Proteína NEDD8 , Poliubiquitina/metabolismo , Proteómica , Especificidad por Sustrato , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
We have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitin chains containing both Lys63-linked and Met1-linked ubiquitin (Ub) oligomers, some of which are attached covalently to Interleukin Receptor Associated kinase 1. Here we show that Lys63/Met1-Ub hybrids are also formed rapidly when the TNFR1/TRADD, TLR3/TRIF- and NOD1/RIP2-signaling networks are activated, some of which are attached covalently to Receptor-Interacting Protein 1 (TNFR1 pathway) or Receptor-Interacting Protein 2 (NOD1 pathway). These observations suggest that the formation of Lys63/Met1-Ub hybrids are of general significance for the regulation of innate immune signaling systems, and their potential roles in vivo are discussed. We also report that TNFα induces the attachment of Met1-linked Ub chains directly to TNF receptor 1, which do not seem to be attached covalently to Lys63-linked or other types of ubiquitin chain.
Asunto(s)
Inmunidad Innata/inmunología , Factores Inmunológicos/inmunología , Lisina/inmunología , Metionina/inmunología , Monocitos/inmunología , Ubiquitina/inmunología , Animales , Células Cultivadas , Humanos , Ratones , Unión Proteica/inmunología , Transducción de Señal/inmunología , Ubiquitinación/inmunologíaRESUMEN
RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7â¼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.
Asunto(s)
Proteínas Portadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cullin/metabolismo , Ciclopentanos/farmacología , Células HEK293 , Humanos , Proteína NEDD8 , Pirimidinas/farmacología , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Ubiquitinas/antagonistas & inhibidores , Ubiquitinas/metabolismoRESUMEN
The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.
Asunto(s)
Reparación del ADN/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Anemia de Fanconi/genética , Animales , Animales Modificados Genéticamente , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Pollos , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Anemia de Fanconi/metabolismo , Recombinación Homóloga , Transducción de Señal/fisiología , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Abnormal regulation of brain glycogen metabolism is believed to underlie insulin-induced hypoglycaemia, which may be serious or fatal in diabetic patients on insulin therapy. A key regulator of glycogen levels is glycogen targeted protein phosphatase 1 (PP1), which dephosphorylates and activates glycogen synthase (GS) leading to an increase in glycogen synthesis. In this study, we show that the gene PPP1R3F expresses a glycogen-binding protein (R3F) of 82.8 kDa, present at the high levels in rodent brain. R3F binds to PP1 through a classical 'RVxF' binding motif and substitution of Phe39 for Ala in this motif abrogates PP1 binding. A hydrophobic domain at the carboxy-terminus of R3F has similarities to the putative membrane binding domain near the carboxy-terminus of striated muscle glycogen targeting subunit G(M)/R(GL), and R3F is shown to bind not only to glycogen but also to membranes. GS interacts with PP1-R3F and is hyperphosphorylated at glycogen synthase kinase-3 sites (Ser640 and Ser644) when bound to R3F(Phe39Ala). Deprivation of glucose or stimulation with adenosine or noradrenaline leads to an increased phosphorylation of PP1-R3F bound GS at Ser640 and Ser644 curtailing glycogen synthesis and facilitating glycogen degradation to provide glucose in astrocytoma cells. Adenosine stimulation also modulates phosphorylation of R3F at Ser14/Ser18.
Asunto(s)
Astrocitoma/enzimología , Neoplasias Encefálicas/enzimología , Proteínas Portadoras/fisiología , Espacio Extracelular/fisiología , Glucosa/farmacología , Glucógeno Sintasa/biosíntesis , Fosfoproteínas Fosfatasas/fisiología , Proteína Fosfatasa 1/fisiología , Transducción de Señal/efectos de los fármacos , Adenosina/farmacología , Agonistas alfa-Adrenérgicos/farmacología , Secuencia de Aminoácidos , Animales , Astrocitoma/genética , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Neoplasias Encefálicas/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , ADN/biosíntesis , ADN/genética , Glucógeno/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis , Norepinefrina/farmacología , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 1/genética , ARN/biosíntesis , ARN/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Silenciador del Gen , Células HEK293 , Humanos , Fenformina/farmacología , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 2C , Interferencia de ARNRESUMEN
Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1-G(L) complex, suppressing glycogen synthesis. Consequently, the interaction of G(L) with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the G(L)-phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the G(L) protein. The resulting G(L)(Y284F/Y284F) mice display hepatic PP1-G(L) activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of G(L) by phosphorylase a operates in vivo. G(L)(Y284F/Y284F) and G(L)(Y284F/+) mice display improved glucose tolerance compared with G(L)(+/+) littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the G(L)-phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the G(L)(Y284F/Y284F) mice lose more body weight and display decreased blood glucose levels in comparison with their G(L)(+/+) littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of G(L) may prevent futile glucose-glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1-G(L) by phosphorylase a in mammals.
Asunto(s)
Glucosa/metabolismo , Glucógeno Fosforilasa de Forma Hepática/metabolismo , Glucógeno Hepático/metabolismo , Proteína Fosfatasa 1/metabolismo , Regulación Alostérica , Animales , Peso Corporal , Cruzamientos Genéticos , Ayuno/sangre , Femenino , Marcación de Gen , Prueba de Tolerancia a la Glucosa , Glucógeno Sintasa/metabolismo , Heterocigoto , Humanos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Fosforilación , Conejos , Pérdida de PesoRESUMEN
A prediabetic phenotype of glucose intolerance, insulin resistance and obesity was observed at approximately 12 months of age in mice homozygous for a null allele of the major skeletal muscle glycogen-targeting subunit G(M) of protein phosphatase 1 (PP1) and derived from a 129/Ola donor strain. In this study, backcrossing of these G(M)-/- mice (termed obese G(M)-/- mice) onto two different genetic backgrounds gave rise to lean, glucose-tolerant, insulin-sensitive G(M)-/- mice (termed lean G(M)-/- mice), indicating that at least one variant gene in the 129/Ola background, not present in the C57BL/6 or 129s2/sV background, is required for the development of the prediabetic phenotype of obese mice. Slightly elevated AMP-activated protein kinase alpha2 activity in the skeletal muscle of lean C57BL/6 mice was also observed to a lesser extent in the obese G(M)-/- mice. Normal or slightly raised in vivo glucose transport in lean C57BL/6 G(M)-/- mice compared with decreased glucose transport in the obese G(M)-/- mice supports the tenet that adequate transport of glucose may be a key factor in preventing the development of the prediabetic phenotype. The pH 6.8/pH 8.6 activity ratio of phosphorylase kinase was increased in lean C57BL/6 G(M)-/- mice compared with controls indicating that phosphorylase kinase is an in vivo substrate of PP1-G(M).
Asunto(s)
Glucógeno/metabolismo , Músculo Esquelético/enzimología , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Quinasas Activadas por AMP , Tejido Adiposo/metabolismo , Animales , Cruzamientos Genéticos , Ambiente , Femenino , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Miembro Posterior/enzimología , Isoenzimas/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/metabolismo , Fenotipo , Proteína Fosfatasa 1/química , Proteínas Serina-Treonina Quinasas/metabolismo , DelgadezRESUMEN
The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.