RESUMEN
The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.
Asunto(s)
Dominio Catalítico , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Simulación de Dinámica Molecular , Conformación Proteica , Regulación Alostérica , Sitio Alostérico , Medición de Intercambio de Deuterio/métodos , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Espectrometría de Masas/métodos , Mutación , Unión ProteicaRESUMEN
Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.
Asunto(s)
Proteínas Repetidas Ricas en Leucina , Proteínas Serina-Treonina Quinasas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Dominios Proteicos , Mutación , Péptidos/metabolismo , FosforilaciónRESUMEN
Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Enfermedad de Parkinson/metabolismo , Anticuerpos de Dominio Único , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Mapeo Epitopo , Células HEK293 , Humanos , Ratones , Microtúbulos/metabolismo , Fosforilación , Unión Proteica , Células RAW 264.7 , Proteínas de Unión al GTP rab/metabolismoRESUMEN
LRRK2 is a multi-domain protein with three catalytically inert N-terminal domains (NtDs) and four C-terminal domains, including a kinase and a GTPase domain. LRRK2 mutations are linked to Parkinson's Disease. Recent structures of LRRK2RCKW and a full-length inactive LRRK2 (fl-LRRK2INACT) monomer revealed that the kinase domain drives LRRK2 activation. The LRR domain and also an ordered LRR- COR linker, wrap around the C-lobe of the kinase domain and sterically block the substrate binding surface in fl-LRRK2INACT. Here we focus on the crosstalk between domains. Our biochemical studies of GTPase and kinase activities of fl-LRRK2 and LRRK2RCKW reveal how mutations influence this crosstalk differently depending on the domain borders investigated. Furthermore, we demonstrate that removing the NtDs leads to altered intramolecular regulation. To further investigate the crosstalk, we used Hydrogen-Deuterium exchange Mass Spectrometry (HDX-MS) to characterize the conformation of LRRK2RCKW and Gaussian Accelerated Molecular Dynamics (GaMD) to create dynamic portraits of fl-LRRK2 and LRRK2RCKW. These models allowed us to investigate the dynamic changes in wild type and mutant LRRK2s. Our data show that the a3ROC helix, the Switch II motif in the ROC domain, and the LRR-ROC linker play crucial roles in mediating local and global conformational changes. We demonstrate how these regions are affected by other domains in fl-LRRK2 and LRRK2RCKW and show how unleashing of the NtDs as well as PD mutations lead to changes in conformation and dynamics of the ROC and kinase domains which ultimately impact kinase and GTPase activities. These allosteric sites are potential therapeutic targets.
RESUMEN
To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Simulación de Dinámica Molecular , Secuencias de Aminoácidos , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Dominios Proteicos , Transporte de ProteínasRESUMEN
Many of the fundamental concepts of signal transduction and kinase activity are attributed to the discovery and crystallization of cAMP-dependent protein kinase, or protein kinase A. PKA is one of the best-studied kinases in human biology, with emphasis in biochemistry and biophysics, all the way to metabolism, hormone action, and gene expression regulation. It is surprising, however, that our understanding of PKA's role in disease is largely underappreciated. Although genetic mutations in the PKA holoenzyme are known to cause diseases such as Carney complex, Cushing syndrome, and acrodysostosis, the story largely stops there. With the recent explosion of genomic medicine, we can finally appreciate the broader role of the Gαs-PKA pathway in disease, with contributions from aberrant functioning G proteins and G protein-coupled receptors, as well as multiple alterations in other pathway components and negative regulators. Together, these represent a broad family of diseases we term the Gαs-PKA pathway signalopathies. The Gαs-PKA pathway signalopathies encompass diseases caused by germline, postzygotic, and somatic mutations in the Gαs-PKA pathway, with largely endocrine and neoplastic phenotypes. Here, we present a signaling-centric review of Gαs-PKA-driven pathophysiology and integrate computational and structural analysis to identify mutational themes commonly exploited by the Gαs-PKA pathway signalopathies. Major mutational themes include hotspot activating mutations in Gαs, encoded by GNAS, and mutations that destabilize the PKA holoenzyme. With this review, we hope to incite further study and ultimately the development of new therapeutic strategies in the treatment of a wide range of human diseases. SIGNIFICANCE STATEMENT: Little recognition is given to the causative role of Gαs-PKA pathway dysregulation in disease, with effects ranging from infectious disease, endocrine syndromes, and many cancers, yet these disparate diseases can all be understood by common genetic themes and biochemical signaling connections. By highlighting these common pathogenic mechanisms and bridging multiple disciplines, important progress can be made toward therapeutic advances in treating Gαs-PKA pathway-driven disease.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , Medicina Genómica , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Mutación , Transducción de SeñalRESUMEN
BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.
Asunto(s)
Insuficiencia Cardíaca , Hipertensión , Células Madre Pluripotentes Inducidas , Humanos , Ratas , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Microtomografía por Rayos X , Células Madre Pluripotentes Inducidas/metabolismo , Hipertensión/complicaciones , Hipertensión/genética , Miocitos Cardíacos/metabolismo , Cardiomegalia , ARNRESUMEN
PRKACA and PRKACB code for two catalytic subunits (Cα and Cß) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cß subunits of PKA during human development.
Asunto(s)
Anomalías Múltiples/genética , Disfunción Cognitiva/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Dedos/anomalías , Mutación de Línea Germinal , Defectos de los Tabiques Cardíacos/genética , Polidactilia/genética , Dedos del Pie/anomalías , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/patología , Adolescente , Adulto , Animales , Secuencia de Bases , Disfunción Cognitiva/diagnóstico , Disfunción Cognitiva/patología , AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/deficiencia , Femenino , Dedos/patología , Regulación del Desarrollo de la Expresión Génica , Defectos de los Tabiques Cardíacos/diagnóstico , Defectos de los Tabiques Cardíacos/patología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Holoenzimas/química , Holoenzimas/deficiencia , Holoenzimas/genética , Humanos , Recién Nacido , Masculino , Ratones , Modelos Moleculares , Mosaicismo , Células 3T3 NIH , Linaje , Polidactilia/diagnóstico , Polidactilia/patología , Estructura Secundaria de Proteína , Dedos del Pie/patologíaRESUMEN
Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cß subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , Inhibidores de Proteínas Quinasas , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/química , Péptidos/químicaRESUMEN
Microtubule-Associated Serine/Threonine (MAST) kinases represent an evolutionary conserved branch of the AGC protein kinase superfamily in the kinome. Since the discovery of the founding member, MAST2, in 1993, three additional family members have been identified in mammals and found to be broadly expressed across various tissues, including the brain, heart, lung, liver, intestine and kidney. The study of MAST kinases is highly relevant for unraveling the molecular basis of a wide range of different human diseases, including breast and liver cancer, myeloma, inflammatory bowel disease, cystic fibrosis and various neuronal disorders. Despite several reports on potential substrates and binding partners of MAST kinases, the molecular mechanisms that would explain their involvement in human diseases remain rather obscure. This review will summarize data on the structure, biochemistry and cell and molecular biology of MAST kinases in the context of biomedical research as well as organismal model systems in order to provide a current profile of this field.
RESUMEN
Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.
Asunto(s)
Degeneración Retiniana , Ratones , Animales , Degeneración Retiniana/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, Cα and Cß, and four functionally nonredundant R-subunits (RIα, RIß, RIIα, RIIß). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, Cß actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the Cα1-subunit, we know almost nothing about these Cß isoforms wherein Cß2 is highly expressed in lymphocytes, and Cß3 and Cß4 isoforms account for â¼50% of PKA signaling in brain. Based on recent disease mutations, the Cß proteins appear to be functionally important and nonredundant with the Cα isoforms. Imaging in retina also supports nonredundant roles for Cß as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the Cα- and Cß-subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , Proteínas Quinasas , Animales , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de SeñalRESUMEN
3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cß proteins are called Cß1, Cß2, Cß3, Cß4 and so-called abc variants of Cß3 and Cß4. Whereas Cß1 is ubiquitously expressed, Cß2 is enriched in immune cells and the Cß3, Cß4 and their abc variants are solely expressed in neuronal cells. All Cα and Cß splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cß splice variants with the exception of Cα1 and Cß1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cß splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies.
Asunto(s)
Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Mutación , Neoplasias/patología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Exones , Humanos , Neoplasias/enzimología , Neoplasias/genética , Homología de Secuencia , Transducción de SeñalRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson's disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a "brake" that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.
Asunto(s)
Dominio Catalítico , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Simulación de Dinámica Molecular , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mutación MissenseRESUMEN
In 1984, Japanese researchers led by the biochemist Hiroyoshi Hidaka described the first synthetic protein kinase inhibitors based on an isoquinoline sulfonamide structure (Hidaka et al. Biochemistry, 1984 Oct 9; 23(21): 5036-41. doi: 10.1021/bi00316a032). These led to the first protein kinase inhibitor approved for medical use (fasudil), an inhibitor of the AGC subfamily Rho kinase. With potencies strong enough to compete against endogenous ATP, the isoquinoline compounds established the druggability of the ATP binding site. Crystal structures of their protein kinase complexes, including with cAMP-dependent protein kinase (PKA), showed interactions that, on the one hand, could mimic ATP but, on the other hand, could be optimized for high potency binding, kinase selectivity, and diversification away from adenosine. They also showed the flexibility of the glycine-rich loop, and PKA became a major prototype for crystallographic and nuclear magnetic resonance (NMR) studies of protein kinase mechanism and dynamic activity control. Since fasudil, more than 70 kinase inhibitors have been approved for clinical use, involving efforts that progressively have introduced new paradigms of data-driven drug discovery. Publicly available data alone comprise over 5000 protein kinase crystal structures and hundreds of thousands of binding data. Now, new methods, including artificial intelligence techniques and expansion of protein kinase targeting approaches, together with the expiration of patent protection for optimized inhibitor scaffolds, promise even greater advances in drug discovery. Looking back to the time of the first isoquinoline hinge binders brings the current state-of-the-art into stark contrast. Appropriately for this Perspective article, many of the milestone papers during this time were published in Biochemistry (now ACS Biochemistry).
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Diseño de Fármacos/historia , Inhibidores de Proteínas Quinasas/farmacología , Adenosina Trifosfato/metabolismo , Inteligencia Artificial , Sitios de Unión/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/ultraestructura , Ciencia de los Datos/historia , Ciencia de los Datos/tendencias , Diseño de Fármacos/métodos , Diseño de Fármacos/tendencias , Descubrimiento de Drogas/historia , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Historia del Siglo XX , Isoquinolinas/química , Isoquinolinas/farmacología , Resonancia Magnética Nuclear Biomolecular , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog-bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive "mixed" intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Regulación Alostérica , Sitios de Unión , GMP Cíclico/análogos & derivados , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Plasmodium falciparum/metabolismo , Dominios Proteicos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Resonancia por Plasmón de SuperficieRESUMEN
Controlled transport of surface-functionalized magnetic beads in a liquid medium is a central requirement for the handling of captured biomolecular targets in microfluidic lab-on-chip biosensors. Here, the influence of the physiological liquid medium on the transport characteristics of functionalized magnetic particles and on the functionality of the coupled protein is studied. These aspects are theoretically modeled and experimentally investigated for prototype superparamagnetic beads, surface-functionalized with green fluorescent protein immersed in buffer solution with different concentrations of a surfactant. The model reports on the tunability of the steady-state particle substrate separation distance to prevent their surface sticking via the choice of surfactant concentration. Experimental and theoretical average velocities are discussed for a ratchet-like particle motion induced by a dynamic external field superposed on a static locally varying magnetic field landscape. The developed model and experiment may serve as a basis for quantitative forecasts on the functionality of magnetic particle transport-based lab-on-chip devices.
Asunto(s)
Técnicas Biosensibles , Tensoactivos , Campos Magnéticos , Magnetismo , MicrofluídicaRESUMEN
Recently, a mutation was discovered in the gene PRKACB encoding the catalytic subunit ß of PKA (PKAcß) from a patient with severe Cushing's syndrome. This mutation, S54L, leads to a structural change in the glycine-rich loop of the protein. In the present study, an inhibitor with six-fold selectivity toward S54L-PKAcß mutant over the wild-type enzyme was constructed. Moreover, we developed a fluorescent assay allowing to determine side by side the affinity of commercially available PKA inhibitors, newly synthesized compounds, and fluorescent probes toward PKAcß and S54L-PKAcß.
Asunto(s)
Adenoma/genética , Adenoma/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/antagonistas & inhibidores , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/química , Hidrocortisona/biosíntesis , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Humanos , MutaciónRESUMEN
Synthetic derivatives of cyclic adenosine monophosphate, such as halogenated or other more hydrophobic analogs, are widely used compounds, to investigate diverse signal transduction pathways of eukaryotic cells. This inspired us to develop cyclic nucleotides, which exhibit chemical structures composed of brominated 7-deazaadenines and the phosphorylated ribosugar. The synthesized 8-bromo- and 7-bromo-7-deazaadenosine-3',5'-cyclic monophosphates rank among the most potent activators of cyclic nucleotide-regulated ion channels as well as cAMP-dependent protein kinase. Moreover, these substances bind tightly to exchange proteins directly activated by cAMP.
Asunto(s)
AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Adenina/análogos & derivados , Adenina/síntesis química , Adenina/química , Adenina/farmacología , Animales , AMP Cíclico/síntesis química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/agonistas , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Activación Enzimática/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/agonistas , Factores de Intercambio de Guanina Nucleótido/metabolismo , Halogenación , Humanos , RatonesRESUMEN
Kinases regulate multiple and diverse signaling pathways and misregulation is implicated in a multitude of diseases. Although significant efforts have been put forth to develop kinase-specific inhibitors, specificity remains a challenge. As an alternative to catalytic inhibition, allosteric inhibitors can target areas on the surface of an enzyme, thereby providing additional target diversity. Using cAMP-dependent protein kinase A (PKA) as a model system, we sought to develop a hydrocarbon-stapled peptide targeting the pseudosubstrate domain of the kinase. A library of peptides was designed from a Protein Kinase Inhibitor (PKI), a naturally encoded protein that serves as a pseudosubstrate inhibitor for PKA. The binding properties of these peptide analogs were characterized by fluorescence polarization and surface plasmon resonance, and two compounds were identified with KD values in the 500-600 pM range. In kinase activity assays, both compounds demonstrated inhibition with 25-35 nM IC50 values. They were also found to permeate cells and localize within the cytoplasm and inhibited PKA activity within the cellular environment. To the best of our knowledge, these stapled peptide inhibitors represent some of the highest affinity binders reported to date for hydrocarbon stapled peptides.