RESUMO
The peptidyl prolyl isomerases (PPI) of the cyclophilin type are distributed throughout human cells, including eight found solely in the nucleus. Nuclear cyclophilins are involved in complexes that regulate chromatin modification, transcription, and pre-mRNA splicing. This review collects what is known about the eight human nuclear cyclophilins: peptidyl prolyl isomerase H (PPIH), peptidyl prolyl isomerase E (PPIE), peptidyl prolyl isomerase-like 1 (PPIL1), peptidyl prolyl isomerase-like 2 (PPIL2), peptidyl prolyl isomerase-like 3 (PPIL3), peptidyl prolyl isomerase G (PPIG), spliceosome-associated protein CWC27 homolog (CWC27), and peptidyl prolyl isomerase domain and WD repeat-containing protein 1 (PPWD1). Each "spliceophilin" is evaluated in relation to the spliceosomal complex in which it has been studied, and current work studying the biological roles of these cyclophilins in the nucleus are discussed. The eight human splicing complexes available in the Protein Data Bank (PDB) are analyzed from the viewpoint of the human spliceophilins. Future directions in structural and cellular biology, and the importance of developing spliceophilin-specific inhibitors, are considered.
Assuntos
Núcleo Celular/química , Ciclofilinas/química , Spliceossomos/química , Relação Estrutura-Atividade , Sequência de Aminoácidos , Ciclofilinas/classificação , Ciclofilinas/genética , Ciclofilinas/metabolismo , Humanos , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Conformação Proteica , Splicing de RNA/genética , Spliceossomos/genéticaRESUMO
Pre-mRNA splicing is a dynamic, multistep process that is catalyzed by the RNA (ribonucleic acid)-protein complex called the spliceosome. The spliceosome contains a core set of RNAs and proteins that are conserved in all organisms that perform splicing. In higher organisms, peptidyl-prolyl isomerase H (PPIH) directly interacts with the core protein pre-mRNA processing factor 4 (PRPF4) and both integrate into the pre-catalytic spliceosome as part of the tri-snRNP (small nuclear RNA-protein complex) subcomplex. As a first step to understand the protein interactions that dictate PPIH and PRPF4 function, we expressed and purified soluble forms of each protein and formed a complex between them. We found two sites of interaction between PPIH and the N-terminus of PRPF4, an unexpected result. The N-terminus of PRPF4 is an intrinsically disordered region and does not adopt secondary structure in the presence of PPIH. In the absence of an atomic resolution structure, we used mutational analysis to identify point mutations that uncouple these two binding sites and find that mutations in both sites are necessary to break up the complex. A discussion of how this bipartite interaction between PPIH and PRPF4 may modulate spliceosomal function is included.
Assuntos
Peptidilprolil Isomerase/metabolismo , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Spliceossomos/metabolismo , Calorimetria , Dicroísmo Circular , Clonagem Molecular , Proteínas Intrinsicamente Desordenadas/metabolismo , Peptidilprolil Isomerase/genética , Ligação Proteica , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ressonância de Plasmônio de Superfície , UltracentrifugaçãoRESUMO
The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Relógios Circadianos , Ritmo Circadiano , Fatores de Transcrição ARNTL/química , Fatores de Transcrição ARNTL/genética , Animais , Linhagem Celular Tumoral , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Humanos , Isomerismo , Camundongos , Mutação , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Filogenia , Prolina , Domínios Proteicos , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Tempo , Transfecção , TriptofanoRESUMO
Developing male germ cells are exquisitely sensitive to environmental insults such as heat and oxidative stress. An additional characteristic of these cells is their unique dependence on RNA-binding proteins for regulating posttranscriptional gene expression and translational control. Here we provide a mechanistic link unifying these two features. We show that the germ cell-specific RNA-binding protein deleted in azoospermia-like (Dazl) is phosphorylated by MAPKAP kinase 2 (MK2), a stress-induced protein kinase activated downstream of p38 MAPK. We demonstrate that phosphorylation of Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein, resulting in reduced translation of Dazl-regulated target RNAs. We further show that transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule, the Drosophila orthologue of human Dazl. These results illuminate a novel role for MK2 in spermatogenesis, expand the repertoire of RNA-binding proteins phosphorylated by this kinase, and suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl.
Assuntos
Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Drosophila/metabolismo , Expressão Gênica , Células Germinativas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA , Espermatogênese/genética , Espermatogênese/fisiologia , Testículo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cyclophilins are ubiquitously expressed proteins that bind to prolines and can catalyse cis/trans isomerization of proline residues. There are 17 annotated members of the cyclophilin family in humans, ubiquitously expressed and localized variously to the cytoplasm, nucleus or mitochondria. Surprisingly, all eight of the nuclear localized cyclophilins are found associated with spliceosomal complexes. However, their particular functions within this context are unknown. We have therefore adapted three established assays for in vitro pre-mRNA splicing to probe the functional roles of nuclear cyclophilins in the context of the human spliceosome. We find that four of the eight spliceosom-associated cyclophilins exert strong effects on splicing in vitro. These effects are dose-dependent and, remarkably, uniquely characteristic of each cyclophilin. Using both qualitative and quantitative means, we show that at least half of the nuclear cyclophilins can act as regulatory factors of spliceosome function in vitro. The present work provides the first quantifiable evidence that nuclear cyclophilins are splicing factors and provides a novel approach for future work into small molecule-based modulation of pre-mRNA splicing.
Assuntos
Ciclofilinas/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA/fisiologia , Spliceossomos/metabolismo , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Ciclofilinas/química , Ciclofilinas/genética , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Precursores de RNA/química , Precursores de RNA/genética , Spliceossomos/química , Spliceossomos/genéticaRESUMO
The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z' factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC(™)) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry-based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway.
Assuntos
Cisteína Endopeptidases/química , Avaliação Pré-Clínica de Medicamentos/métodos , Recuperação de Fluorescência Após Fotodegradação/métodos , Corantes Fluorescentes/química , Espectrometria de Massas/métodos , Proteínas Relacionadas à Autofagia , Cisteína Endopeptidases/análise , Ativação Enzimática , Especificidade por SubstratoRESUMO
Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity.
Assuntos
Ciclofilinas/química , Ciclofilinas/metabolismo , Família Multigênica , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand- and receptor-expressing cells. Consequently, the ephrins and ephrin receptor tyrosine kinases often mediate processes involving cell-cell contact, including cellular adhesion or repulsion, developmental remodeling and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small-cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and is suitable for co-crystallization studies. Complex structures show multiple contacts between kinase and substrates; in particular, two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes.
Assuntos
Efrinas/química , Receptores Proteína Tirosina Quinases/química , Cristalografia por Raios X , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA3 , Especificidade por SubstratoRESUMO
Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.
Assuntos
Lipase/química , Lipídeos/química , Animais , Inibidores da Colinesterase/química , Cristalografia por Raios X , Glicosilação , Humanos , Cinética , Lipase/genética , Lipase/metabolismo , Lipídeos/genética , Paraoxon/química , Estrutura Terciária de Proteína/fisiologia , RatosRESUMO
Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamembrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes.
Assuntos
Receptores Proteína Tirosina Quinases/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Homeostase , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA3 , Homologia de Sequência de Aminoácidos , Tirosina/metabolismoRESUMO
Cyclophilins comprise one of the three classes of peptidylprolyl isomerases found in all eukaryotic and prokaryotic organisms, as well as viruses. Many of the 17 annotated human cyclophilins contain the catalytic domain in tandem with other domains, and many of the specific functions of a particular cyclophilin or its associated domains remain unknown. The structure of the isomerase domain from a spliceosome-associated cyclophilin, PPWD1 (peptidylprolyl isomerase containing WD40 repeat), has been solved to 1.65 A. In the crystal, the N-terminus of one isomerase domain is bound in the active site of a neighboring isomerase molecule in a manner analogous to substrate. NMR solution studies show that this sequence binds to the active site of the cyclophilin, but cannot be turned over by the enzyme. A pseudo-substrate immediately N-terminal to the cyclophilin domain in PPWD1 could have wider implications for the function of this cyclophilin in the spliceosome, where it is located in human cells.
Assuntos
Peptidilprolil Isomerase/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ciclofilinas/química , Ciclofilinas/classificação , Ciclofilinas/metabolismo , Humanos , Ligação de Hidrogênio , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptidilprolil Isomerase/genética , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Análise Espectral Raman , Spliceossomos/química , Spliceossomos/metabolismo , Água/químicaRESUMO
Calpains are calcium activated cysteine proteases found throughout the animal, plant, and fungi kingdoms; 14 isoforms have been described in the human genome. Calpains have been implicated in multiple models of human disease; for instance, calpain 1 is activated in the brains of individuals with Alzheimer's disease, and the digestive tract specific calpain 9 is down-regulated in gastric cancer cell lines. We have solved the structures of human calpain 1 and calpain 9 protease cores using crystallographic methods; both structures have clear implications for the function of non-catalytic domains of full-length calpains in the calcium-mediated activation of the enzyme. The structure of minicalpain 1 is similar to previously solved structures of the protease core. Auto-inhibition in this system is most likely through rearrangements of a central helical/loop region near the active site cysteine, which occlude the substrate binding site. However, the structure of minicalpain 9 indicates that auto-inhibition in this enzyme is mediated through large intra-domain movements that misalign the catalytic triad. This disruption is reminiscent of the full-length inactive calpain conformation. The structures of the highly conserved, ubiquitously expressed human calpain 1 and the more tissue specific human calpain 9 indicate that although there are high levels of sequence conservation throughout the calpain family, isolated structures of family members are insufficient to explain the molecular mechanism of activation for this group of proteins.
Assuntos
Calpaína/química , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
G protein betagamma subunits associate with many binding partners in cellular signaling cascades. In previous work, we used random-peptide phage display screening to identify a diverse family of peptides that bound to a common surface on Gbetagamma subunits and blocked a subset of Gbetagamma effectors. Later studies showed that one of the peptides caused G protein activation through a novel Gbetagamma-dependent, nucleotide exchange-independent mechanism. Here we report the X-ray crystal structure of Gbeta(1)gamma(2) bound to this peptide, SIGK (SIGKAFKILGYPDYD), at 2.7 A resolution. SIGK forms a helical structure that binds the same face of Gbeta(1) as the switch II region of Galpha. The interaction interface can be subdivided into polar and nonpolar interfaces that together contain a mixture of binding determinants that may be responsible for the ability of this surface to recognize multiple protein partners. Systematic mutagenic analysis of the peptide-Gbeta(1) interface indicates that distinct sets of amino acids within this interface are required for binding of different peptides. Among these unique amino acid interactions, specific electrostatic binding contacts within the polar interface are required for peptide-mediated subunit dissociation. The data provide a mechanistic basis for multiple target recognition by Gbetagamma subunits with diverse functional interactions within a common interface and suggest that pharmacological targeting of distinct regions within this interface could allow for selective manipulation of Gbetagamma-dependent signaling pathways.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Mapeamento de Interação de Proteínas , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Secundária de Proteína , Propriedades de SuperfícieRESUMO
Skin irritation due to iontophoresis may limit the frequency of use of devices for drug delivery or transdermal extraction of analytes of clinical interest. This study examined whether preapplication of corticosteroid preparations could reduce skin irritation from iontophoresis used by the GlucoWatch G2 Biographer (Cygnus, Inc., Redwood City, CA) in monitoring interstitial glucose levels frequently and automatically. Numerous corticosteroid preparations were screened to identify formulations that did not interfere with adhesion of the Biographer to the skin or glucose sensing. Kenalog (Westwood-Squibb Pharmaceuticals, Inc., Buffalo, NY) (triamcinolone acetonide) and Cortizone-10 Quick Shot (Pfizer, Inc., New York, NY) (hydrocortisone) sprays were selected and, in a double-masked, randomized, controlled trial, were applied to the forearms of 66 subjects with diabetes and allowed to dry. Biographers were applied and worn for 15 h, and home blood glucose measurements were taken every 30 min to assess accuracy. Irritation was assessed periodically by trained observers and study subjects. Skin irritation was reduced by both corticosteroid sprays, with the fraction of subjects who experienced moderate irritation reduced by 57% and 43% for the Kenalog and Cortizone-10 Quick Shot sprays, respectively. The treatment effect persisted at the 1-week assessment. Preapplication of these preparations did not affect the clinical utility of interstitial glucose readings. Preapplication of Kenalog or Cortizone-10 Quick Shot sprays significantly reduced skin irritation due to iontophoresis, and did not interfere with glucose measurements. This approach may enable the minority of users who experience moderate to severe skin irritation to use the Biographer more frequently for diabetes management.
Assuntos
Corticosteroides/uso terapêutico , Glicemia/análise , Monitorização Ambulatorial/métodos , Dermatopatias/sangue , Dermatopatias/tratamento farmacológico , Edema/sangue , Edema/tratamento farmacológico , Eritema/sangue , Eritema/tratamento farmacológico , Humanos , Monitorização Ambulatorial/instrumentaçãoRESUMO
OBJECTIVE: This study was done to evaluate the accuracy and safety of measuring glucose with the GlucoWatch biographer in children and adolescents with diabetes. METHODS: Accuracy was assessed by comparing biographer glucose measurements with hourly blood glucose measurements using the HemoCue (Aktiebolaget Leo, Helsingborg, Sweden) Photometer for up to 12 h of monitoring. Safety was evaluated by examining the biographer application sites immediately upon removal of the devices, and then at regular intervals. RESULTS: Sixty-six subjects each wore three biographers at sites including the forearm, upper arm, leg, and torso. For forearm biographers, the mean absolute relative difference between biographer readings and blood glucose was 21%. Ninety-five per cent of biographer readings fell into the A or B regions of the Clarke error grid, and 97.3% into the A or B regions of the consensus error grid. Data from biographers worn at the alternative sites were similar to data from the forearm biographers. Two strong reactions to the adhesive pad of the biographer AutoSensor were observed. Most skin reactions were mild. CONCLUSIONS: The GlucoWatch biographer is well tolerated by children and adolescents with diabetes. Performance is similar when the device is worn at different anatomical sites, and is similar to the performance on the forearm, previously reported in adults.