RESUMEN
Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , VIH-1/crecimiento & desarrollo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Adulto , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Citocinas/metabolismo , Femenino , Expresión Génica/genética , Infecciones por VIH/inmunología , VIH-1/genética , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Cultivo Primario de Células , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/metabolismo , Células Th17/fisiología , Factores de Transcripción/metabolismo , Viremia/inmunología , Viremia/virología , Replicación Viral/fisiologíaRESUMEN
Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide hormone secreted by the pancreatic ß-cells. IAPP plays a role in glycemic regulation, but in the pre-type-2 diabetic state, it aggregates to form an islet amyloid. The process of islet amyloid formation contributes to ß-cell dysfunction and disease progression. The features of the IAPP sequence that modulate amyloid formation are still not understood. Human IAPP contains three aromatic residues, F15, F23, and Y37. F15 and Y37 are highly conserved, while F23 is more commonly a Leu or Ile in other species. The role of the aromatic residues in modulating the time course of amyloid formation and the cytotoxicity was examined using aromatic to Leu mutations. All three single and double mutants and the triple mutant were studied. F23 plays a dominant role in both amyloid formation and toxicity. An F15L mutant accelerated amyloid formation, a Y37L mutant had little effect, while an F23L replacement slowed amyloid formation by a factor of 2.6. Double mutants, which contained an F23L replacement, had a larger effect than those that did not, and there are non-additive effects between pairs of aromatic residues. F23 also plays a key role in toxicity. Single or multiple mutants that contain the F23L replacement were noticeably less toxic than the wild-type or mutants which did not include the F23L substitution. In contrast, the F15L mutant was more toxic than the wild-type one. The implications for IAPP amyloid formation and for the design of non-aggregating analogues of IAPP are discussed.
Asunto(s)
Amiloidosis , Células Secretoras de Insulina , Humanos , Secuencia de Aminoácidos , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Polipéptido Amiloide de los Islotes Pancreáticos/química , Amiloide/genética , Amiloidosis/genéticaRESUMEN
Human islet amyloid polypeptide (hIAPP) plays a role in glucose regulation but forms pancreatic amyloid deposits in type 2 diabetes, and that process contributes to ß-cell dysfunction. Not all species develop diabetes, and not all secrete an IAPP that is amyloidogenic in vitro under normal conditions, a perfect correlation currently exists between both. Studies of IAPPs from such organisms can provide clues about the high amyloidogenicity of hIAPP and can inform the design of soluble analogues of hIAPP. Sheep and goat IAPP are among the most divergent from hIAPP, with 13 and 11 substitutions, respectively, including an unusual Tyr to His substitution at the C-terminus. The properties of sheep and goat IAPP were examined in solution and in the presence of anionic vesicles, resulting in no observed amyloid formation, even at increased concentrations. Furthermore, both peptides are considerably less toxic to cultured ß-cells than hIAPP. The effect of the Y37H replacements was studied in the context of hIAPP, as was a Y37R substitution. Buffer- and salt-dependent effects were observed. There was little impact on the time to form amyloid in phosphate-buffered saline; however, a significant deceleration was observed in Tris buffer, and amyloid formation was slower in the absence of added salt. The Y37H substitution had little impact on toxicity, while the Y37R replacement led to a 30% decrease in toxicity compared with that of hIAPP. The implications for the amyloidogenicity of hIAPP and the design of soluble analogues of the human peptide are discussed.
Asunto(s)
Amiloidosis , Diabetes Mellitus Tipo 2 , Humanos , Ovinos , Animales , Polipéptido Amiloide de los Islotes Pancreáticos/química , Cabras , Amiloide/químicaRESUMEN
The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic ß-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured ß-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured ß-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation.
Asunto(s)
Diabetes Mellitus Tipo 2 , Polipéptido Amiloide de los Islotes Pancreáticos , Secuencia de Aminoácidos , Amiloide/toxicidad , Animales , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , PapioRESUMEN
Amyloid formation by amylin contributes to ß-cell dysfunction in type 2 diabetes. The features that control the amyloidogenicity and toxicity of amylin are not understood. Not all species form islet amyloid, and its presence or absence correlates with the in vitro behavior of the polypeptide. Rats do not develop type 2 diabetes or islet amyloid, and rat amylin is non-amyloidogenic, except at very high concentrations. This has led to the notion that rodent amylins are non-amyloidogenic. Prairie vole amylin has an unusual sequence compared to those of human and rat amylin, including nonconservative Lys-1 to Glu and Asn-22 to Gly substitutions. The first reduces the net charge on the peptide, while the second disrupts a potential network of side chain hydrogen bonds in the amyloid fiber, a so-called Asn ladder. The prairie vole polypeptide forms amyloid more slowly than human amylin and is considerably less cytotoxic. An Asn-22 to Gly substitution in human amylin significantly reduces toxicity, increasing the effective concentration of amylin required to reach 50% toxicity by >7-fold, but has modest effects on the time to form amyloid. A Lys-1 to Glu replacement has a weaker effect but does reduce toxicity relative to that of human amylin, without having a significant impact on the time to form amyloid. The effect of the Lys-1 to Glu substitution on amyloid kinetics is more significant in Tris buffer than in phosphate-buffered saline. This work demonstrates that the N-terminus of amylin plays a role in modulating toxicity and highlights the key role of position 22.
Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Secuencia de Aminoácidos/genética , Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , Proteínas Amiloidogénicas/toxicidad , Amiloidosis/genética , Amiloidosis/metabolismo , Animales , Arvicolinae , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , Cinética , Ratas , Alineación de Secuencia/métodosRESUMEN
Pancreatic amyloid formation by the polypeptide IAPP contributes to ß-cell dysfunction in type 2 diabetes. There is a 1:1 correspondence between the ability of IAPP from different species to form amyloid in vitro and the susceptibility of the organism to develop diabetes. Rat IAPP is non-amyloidogenic and differs from human IAPP at six positions, including three proline replacements: A25P, S28P, and S29P. Incorporation of these proline residues into human IAPP leads to a non-amyloidogenic analogue that is used clinically. The role of the individual proline residues is not understood. We examine the three single and three double proline substitutions in the context of human IAPP. An S28P substitution significantly decreases amyloidogenicity and toxicity, while an S29P substitution has very modest effects despite being an identical replacement just one residue away. The consequences of the A25P substitution are between those of the two Ser to Pro substitutions. Double analogues containing an S28P replacement are less amyloidogenic and less toxic than the IAPPA25P S29P double analogue. Ion mobility mass spectrometry reveals that there is no correlation between the monomer or dimer conformation as reported by collision cross section measurements and the time to form amyloid. The work reveals both the plasticity of IAPP amyloid formation and the exquisite sequence sensitivity of IAPP amyloidogenicity and toxicity. The study highlights the key role of the S28P substitution and provides information that will aid in the rational design of soluble variants of IAPP. The variants studied here offer a system for further exploring features that control IAPP toxicity.
Asunto(s)
Algoritmos , Sustitución de Aminoácidos/genética , Amiloide/genética , Variación Genética/genética , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Prolina/genética , Secuencia de Aminoácidos , Amiloide/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Prolina/metabolismoRESUMEN
Dictyostelium discoideum (D. discoideum) is a simple eukaryote with a unique life cycle in which it differentiates from unicellular amoebae into a fruiting body upon starvation. Reactive oxygen species (ROS) have been associated with bacterial predation, as well as regulatory events during D. discoideum development and differentiation. Coenzyme A (CoA) is a key metabolic integrator in all living cells. A novel function of CoA in redox regulation, mediated by covalent attachment of CoA to cellular proteins in response to oxidative or metabolic stress, has been recently discovered and termed protein CoAlation. In this study, we report that the level of CoA and protein CoAlation in D. discoideum are developmentally regulated, and correlate with the temporal expression pattern of genes implicated in CoA biosynthesis during morphogenesis. Furthermore, treatment of growing D. discoideum cells with oxidising agents results in a dose-dependent increase of protein CoAlation. However, much higher concentrations were required when compared to mammalian cells and bacteria. Increased resistance of D. discoideum to oxidative stress induced by H2O2 has previously been attributed to high levels of catalase activity. In support of this notion, we found that H2O2-induced protein CoAlation is significantly increased in CatA-deficient D. discoideum cells. Collectively, this study provides insights into the role of CoA and protein CoAlation in the maintenance of redox homeostasis in amoeba and during D. discoideum morphogenesis.
Asunto(s)
Coenzima A/metabolismo , Dictyostelium/crecimiento & desarrollo , Estrés Oxidativo , Proteínas Protozoarias/metabolismo , Dictyostelium/citología , Dictyostelium/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Morfogénesis , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Infecciones por Protozoos/parasitología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/inmunología , Digoxina/farmacología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Integración Viral/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Activación de Linfocitos/efectos de los fármacos , Latencia del Virus/efectos de los fármacosRESUMEN
In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress. In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.
Asunto(s)
Antioxidantes/metabolismo , Proteínas Bacterianas/metabolismo , Coenzima A/metabolismo , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Coenzima A/genética , Diamida/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Oxidación-Reducción , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genéticaRESUMEN
Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.
Asunto(s)
Coenzima A/metabolismo , Proteínas/metabolismo , Animales , Cisteína/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Riñón/metabolismo , Hígado/metabolismo , Masculino , Miocardio/metabolismo , Especificidad de Órganos , Oxidación-Reducción , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Conejos , Ratas Sprague-Dawley , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Latency allows HIV-1 to persist in long-lived cellular reservoirs, preventing virus eradication. We have previously shown that the heat shock protein 90 (Hsp90) is required for HIV-1 gene expression and mediates greater HIV-1 replication in conditions of hyperthermia. Here we report that specific inhibitors of Hsp90 such as 17-(N-allylamino)-17-demethoxygeldanamycin and AUY922 prevent HIV-1 reactivation in CD4+ T cells. A single modification at position 19 in the Hsp90 inhibitors abolished this activity, supporting the specificity of the target. We tested the impact of Hsp90 on known pathways involved in HIV-1 reactivation from latency; they include protein kinase Cs(PKCs), mitogen activated protein kinase/extracellular signal regulated kinase/positive transcriptional elongation factor-b and NF-κB. We found that Hsp90 was required downstream of PKCs and was not required for mitogen activated protein kinase activation. Inhibition of Hsp90 reduced degradation of IkBα and blocked nuclear translocation of transcription factor p65/p50, suppressing the NF-κB pathway. Coimmunoprecipitation experiments showed that Hsp90 interacts with inhibitor of nuclear factor kappa-B kinase (IKK) together with cochaperone Cdc37, which is critical for the activity of several kinases. Targeting of Hsp90 by AUY922 dissociated Cdc37 from the complex. Therefore, Hsp90 controls HIV-1 reactivation from latency by keeping the IKK complex functional and thus connects T-cell activation with HIV-1 replication. AUY922 is in phase II clinical trial and, in combination with a PKC-Ï inhibitor in phase II clinical trial, almost completely suppressed HIV-1 reactivation at 15 nM with no cytotoxicity. Selective targeting of the Hsp90/Cdc37 interaction may provide a powerful approach to suppress HIV-1 reactivation from latency.
Asunto(s)
VIH-1/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Activación Viral/fisiología , Latencia del Virus/fisiología , Western Blotting , Línea Celular , VIH-1/metabolismo , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Transducción de Señal/genéticaRESUMEN
The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to ß-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO-) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured ß-cells, although hAmylin-COO- is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132-49 (2018)) shows that Amylin- COO- exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO- exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.
Asunto(s)
Diabetes Mellitus Tipo 2 , Hormonas Peptídicas , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Diabetes Mellitus Tipo 2/metabolismo , Amidas , Proteínas Amiloidogénicas , Amiloide/químicaRESUMEN
Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin's primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.
Asunto(s)
Amiloide , Polipéptido Amiloide de los Islotes Pancreáticos , Humanos , Ratas , Animales , Receptores de Polipéptido Amiloide de Islotes PancreáticosRESUMEN
Amyloids are partially ordered, proteinaceous, ß-sheet rich deposits that have been implicated in a wide range of diseases. An even larger set of proteins that do not normally form amyloid in vivo can be induced to do so in vitro. A growing number of structures of amyloid fibrils have been reported and a common feature is the presence of a tightly packed core region in which adjacent monomers pack together in extremely tight interfaces, often referred to as steric zippers. A second common feature of many amyloid fibrils is their polymorphous nature. We examine the consequences of disrupting the tight packing in amyloid fibrils on the kinetics of their formation using the 37 residue polypeptide hormone islet amyloid polypeptide (IAPP, amylin) as a model system. IAPP forms islet amyloid in vivo and is aggressively amyloidogenic in vitro. Six Cryo-EM structures of IAPP amyloid fibrils are available and in all Gly24 is in the core of the structured region and makes tight contacts with other residues. Calculations using the ff14SBonlysc forcefield in Amber20 show that substitutions with larger amino acids significantly disrupt close packing and are predicted to destabilize the various fibril structures. However, Gly to 2-amino butyric acid (2-carbon side chain) and Gly to Leu substitutions actually enhance the rate of amyloid formation. A Pro substitution slows, but does not prevent amyloid formation.
Asunto(s)
Amiloide , Polipéptido Amiloide de los Islotes Pancreáticos , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Polipéptido Amiloide de los Islotes Pancreáticos/química , Amiloide/químicaRESUMEN
The neuropancreatic polypeptide hormone amylin forms pancreatic islet amyloid in type-2 diabetes. Islet amyloid formation contributes to ß-cell death in the disease and to the failure of islet transplants, but the features which influence amylin amyloidogenicity are not understood. We constructed an amino acid sequence alignment of 202 sequences of amylin and used the alignment to design consensus sequences of vertebrate amylins, mammalian amylins, and primate amylins. Amylin is highly conserved, but there are differences between human amylin and each consensus sequence, ranging from one to six substitutions. Biophysical analysis shows that all of the consensus sequences form amyloid but do so more slowly than human amylin in vitro. The rate of amyloid formation by the primate consensus sequence is 3- to 4-fold slower than human amylin; the mammalian consensus sequence is approximately 20- to 25-fold slower, and the vertebrate consensus sequence is approximately 6-fold slower. All of the consensus sequences are moderately less toxic than human amylin toward a cultured ß-cell line, with the vertebrate consensus sequence displaying the largest reduction in toxicity of 3- to 4-fold. All of the consensus sequences activate a human amylin receptor and exhibit only modest reductions in activity, ranging from 3- to 4-fold as judged by a cAMP production assay. The analysis argues that there is no strong selective evolutionary pressure to avoid the formation of islet amyloid and provides information relevant to the design of less amyloidogenic amylin variants.
Asunto(s)
Amiloide/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Secuencia de Aminoácidos , Amiloide/química , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Secuencia de Consenso , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Modelos Moleculares , Ratas , Alineación de SecuenciaRESUMEN
Spores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores' metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.
Asunto(s)
Bacillus/metabolismo , Coenzima A/metabolismo , Cisteína/metabolismo , Esporas Bacterianas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Bacillus/citología , Proteínas Bacterianas/metabolismo , Disulfuros/química , Disulfuros/metabolismoRESUMEN
Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a "dual anchor" mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.
Asunto(s)
Aurora Quinasa A/química , Aurora Quinasa A/metabolismo , Coenzima A/administración & dosificación , Animales , Coenzima A/química , Coenzima A/farmacología , Cristalografía por Rayos X , Células HEK293 , Células Hep G2 , Humanos , Ratones , Modelos Moleculares , Estrés Oxidativo , Fosforilación , Conformación Proteica , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismoRESUMEN
The polypeptide amylin is responsible for islet amyloid in type 2 diabetes, a process which contributes to ß-cell death in the disease. The role of the N-terminal region of amylin in amyloid formation is relatively unexplored, although removal of the disulfide bridged loop between Cys-2 and Cys-7 accelerates amyloid formation. We examine the des Lys-1 variant of human amylin (h-amylin), a variant which is likely produced in vivo. Lys-1 is a region of high charge density in the h-amylin amyloid fiber. The des Lys-1 polypeptide forms amyloid on the same time scale as wild-type amylin in phosphate buffered saline, but does so more rapidly in Tris. The des Lys-1 variant is somewhat less toxic to cultured INS cells than wild type. The implications for the in vitro mechanism of amyloid formation and for comparative analysis of amyloidogenicity are discussed.
Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , Agregado de Proteínas , Secuencia de Aminoácidos , Tampones (Química) , Línea Celular , Humanos , CinéticaRESUMEN
The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer's Aß peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aß also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aß amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aß amyloid formation and inhibit Aß induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured ß-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aß and h-amylin amyloid formation, illustrates the limitation of using Aß inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.
Asunto(s)
Agonistas de los Receptores de Amilina/farmacología , Amiloide/antagonistas & inhibidores , Bioensayo/normas , Diseño de Fármacos , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Colorantes de Rosanilina/farmacología , Compuestos de Tritilo/farmacología , Amiloide/metabolismo , Naftalenosulfonatos de Anilina/farmacología , Benzotiazoles/farmacología , Colorantes Fluorescentes/farmacología , Humanos , Indicadores y Reactivos/farmacologíaRESUMEN
The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.