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1.
Electrophoresis ; 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233206

RESUMO

Charge heterogeneity analysis of monoclonal antibodies (mAbs) and complex formats, such as bispecifics, is crucial for therapeutic applications. In this study, we developed two capillary electrophoresis (CE)-based methods, capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF), for analyzing a broad spectrum of mAbs and complex mAb formats. For CZE, we introduced a new buffer system and optimized the background electrolyte (BGE) with an alternative dynamic coating agent and a superior polymeric additive. The pH of the BGE was increased, leading to enhanced resolution of high pI and complex format mAbs. In iCIEF, we identified an ampholyte combination offering a highly linear pH gradient and covering a suitable pH range. We also investigated alternatives to denaturing stabilizers and found that non-detergent sulfobetaine 195 exhibited excellent properties for iCIEF applications. These optimized methods provide a framework for the charge heterogeneity analysis of therapeutic mAbs and complex formats.

2.
Phys Chem Chem Phys ; 22(42): 24359-24364, 2020 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-33084665

RESUMO

The knowledge of thermodynamic properties for novel electrolyte formulations is of fundamental interest for industrial applications as well as academic research. Herewith, we present an artificial neural networks (ANN) approach for the prediction of solvation energies and entropies for distinct ion pairs in various protic and aprotic solvents. The considered feed-forward ANN is trained either by experimental data or computational results from conceptual density functional theory calculations. The proposed concept of mapping computed values to experimental data lowers the amount of time-consuming and costly experiments and helps to overcome certain limitations. Our findings reveal high correlation coefficients between predicted and experimental values which demonstrate the validity of our approach.

3.
Cells ; 9(6)2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32630525

RESUMO

cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to ß-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.


Assuntos
Proteínas Quinases/metabolismo , Serina/metabolismo , Treonina/metabolismo , Humanos , Transdução de Sinais
4.
Proc Natl Acad Sci U S A ; 116(30): 14979-14988, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31292254

RESUMO

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.


Assuntos
Domínio Catalítico , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Simulação de Dinâmica Molecular , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação de Sentido Incorreto
5.
Molecules ; 24(8)2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31009996

RESUMO

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.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/química , Peptídeos/química , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Sítios de Ligação , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Ressonância de Plasmônio de Superfície
6.
J Cell Biol ; 217(6): 2167-2184, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29615473

RESUMO

Type II isoforms of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA-II) contain a phosphorylatable epitope within the inhibitory domain of RII subunits (pRII) with still unclear function. In vitro, RII phosphorylation occurs in the absence of cAMP, whereas staining of cells with pRII-specific antibodies revealed a cAMP-dependent pattern. In sensory neurons, we found that increased pRII immunoreactivity reflects increased accessibility of the already phosphorylated RII epitope during cAMP-induced opening of the tetrameric RII2:C2 holoenzyme. Accordingly, induction of pRII by cAMP was sensitive to novel inhibitors of dissociation, whereas blocking catalytic activity was ineffective. Also in vitro, cAMP increased the binding of pRII antibodies to RII2:C2 holoenzymes. Identification of an antibody specific for the glycine-rich loop of catalytic subunits facing the pRII-epitope confirmed activity-dependent binding with similar kinetics, proving that the reassociation is rapid and precisely controlled. Mechanistic modeling further supported that RII phosphorylation precedes cAMP binding and controls the inactivation by modulating the reassociation involving the coordinated action of phosphodiesterases and phosphatases.


Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Subunidades Proteicas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos/farmacologia , Extratos Celulares , Permeabilidade da Membrana Celular/efeitos dos fármacos , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Isoenzimas/metabolismo , Masculino , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
JCI Insight ; 3(8)2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29669941

RESUMO

Mutations in the gene encoding the protein kinase A (PKA) catalytic subunit α have been found to be responsible for cortisol-producing adenomas (CPAs). In this study, we identified by whole-exome sequencing the somatic mutation p.S54L in the PRKACB gene, encoding the catalytic subunit ß (Cß) of PKA, in a CPA from a patient with severe Cushing syndrome. Bioluminescence resonance energy transfer and surface plasmon resonance assays revealed that the mutation hampers formation of type I holoenzymes and that these holoenzymes were highly sensitive to cAMP. PKA activity, measured both in cell lysates and with recombinant proteins, based on phosphorylation of a synthetic substrate, was higher under basal conditions for the mutant enzyme compared with the WT, while maximal activity was lower. These data suggest that at baseline the PRKACB p.S54L mutant drove the adenoma cells to higher cAMP signaling activity, probably contributing to their autonomous growth. Although the role of PRKACB in tumorigenesis has been suggested, we demonstrated for the first time to our knowledge that a PRKACB mutation can lead to an adrenal tumor. Moreover, this observation describes another mechanism of PKA pathway activation in CPAs and highlights the particular role of residue Ser54 for the function of PKA.


Assuntos
Adenoma/enzimologia , Síndrome de Cushing/diagnóstico por imagem , Proteínas Quinases Dependentes de AMP Cíclico/genética , Hidrocortisona/metabolismo , Adenoma/metabolismo , Neoplasias das Glândulas Suprarrenais/diagnóstico por imagem , Neoplasias das Glândulas Suprarrenais/patologia , Neoplasias das Glândulas Suprarrenais/cirurgia , Insuficiência Adrenal/etiologia , Adrenalectomia/métodos , Adulto , Domínio Catalítico/genética , Síndrome de Cushing/patologia , Síndrome de Cushing/cirurgia , Proteína Receptora de AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico , Feminino , Holoenzimas/metabolismo , Humanos , Mutação , Resultado do Tratamento , Sequenciamento do Exoma/métodos
8.
ACS Infect Dis ; 4(3): 415-423, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29251493

RESUMO

cGMP-dependent protein kinase from Plasmodium falciparum ( PfPKG) plays a crucial role in the sexual as well as the asexual proliferation of this human malaria causing parasite. However, function and regulation of PfPKG are largely unknown. Previous studies showed that the domain organization of PfPKG significantly differs from human PKG ( hPKG) and indicated a critical role of the cyclic nucleotide binding domain D (CNB-D). We identified a novel mechanism, where the CNB-D controls activation and regulation of the parasite specific protein kinase. Here, kinase activity is not dependent on a pseudosubstrate autoinhibitory sequence (IS), as reported for human PKG. A construct lacking the putative IS and containing only the CNB-D and the catalytic domain is inactive in the absence of cGMP and can efficiently be activated with cGMP. On the basis of structural evidence, we describe a regulatory mechanism, whereby cGMP binding to CNB-D induces a conformational change involving the αC-helix of the CNB-D. The inactive state is defined by a unique interaction between Asp597 of the catalytic domain and Arg528 of the αC-helix. The same arginine (R528), however, stabilizes cGMP binding by interacting with Tyr480 of the phosphate binding cassette (PBC). This represents the active state of PfPKG. Our results unveil fundamental differences in the activation mechanism between PfPKG and hPKG, building the basis for the development of strategies for targeted drug design in fighting malaria.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Plasmodium falciparum/enzimologia , Regulação Alostérica , GMP Cíclico/metabolismo , Ligação Proteica , Conformação Proteica
9.
Metallomics ; 9(11): 1576-1584, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29043344

RESUMO

Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloenzymes they employ divalent cations for catalysis and regulation. We used the catalytic (C) subunit of cAMP-dependent protein kinase (PKA) as a model protein to investigate the role of a variety of physiologically or pathophysiologically relevant divalent metal ions in distinct steps within the catalytic cycle. It is established that divalent metal ions play a crucial role in co-binding of nucleotides and also assist in catalysis. Our studies reveal that besides the physiologically highly relevant magnesium, metals like zinc and manganese can assist in phosphoryl transfer, however, only a few support efficient substrate turnover (turnover catalysis). Those trace metals allow for substrate binding and phosphotransfer but hamper product release. We further established the unique role of magnesium as the common biologically relevant divalent metal ideally enabling (co-) substrate binding and orientation. Magnesium allows stable substrate binding and, on the other hand accelerates product release, thus being extremely efficient in turnover catalysis. We extended our studies to non-catalytic functions of protein kinases looking at pseudokinases, a subfamily of protein kinases inherently lacking critical residues for catalysis. Recently, pseudokinases have been linked to human diseases. Some pseudokinases are still capable of binding metal ions, yet have lost the ability to transfer the phosphoryl group from ATP to a given substrate. Here metal ions stabilize an active like, though catalytically unproductive conformation and are therefore crucial to maintain non-catalytic function. Finally, we demonstrate for the canonical kinase PKA that the trace metal manganese alone can stabilize protein kinases in an active like conformation allowing them to bind substrates even in the absence of nucleotides.


Assuntos
Cátions Bivalentes/farmacologia , Ensaios Enzimáticos/métodos , Metais/farmacologia , Proteínas Quinases/metabolismo , Biocatálise/efeitos dos fármacos , Cádmio/farmacologia , Cálcio/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Magnésio/farmacologia , Manganês/farmacologia , Nucleotídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Zinco/farmacologia
10.
Biochem Soc Trans ; 45(3): 653-663, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28620027

RESUMO

Protein phosphorylation, mediated by protein kinases, is a key event in the regulation of eukaryotic signal transduction. The majority of eukaryotic protein kinases perform phosphoryl transfer, assisted by two divalent metal ions. About 10% of all human protein kinases are, however, thought to be catalytically inactive. These kinases lack conserved residues of the kinase core and are classified as pseudokinases. Yet, it has been demonstrated that pseudokinases are critically involved in biological functions. Here, we show how pseudokinases have developed strategies by modifying amino acid residues in order to achieve stable, active-like conformations. This includes binding of the co-substrate ATP in a two metal-, one metal- or even no metal-binding mode. Examples of the respective pseudokinases are provided on a structural basis and compared with a canonical protein kinase, Protein Kinase A. Moreover, the functional roles of both independent metal-binding sites, Me1 and Me2, are discussed. Lack of phosphotransferase activity does not implicate a loss of function and can easily point to alternative roles of pseudokinases, i.e. acting as switches or scaffolds, and having evolved as components crucial for cellular cross-talk and signaling. Interestingly, pseudokinases are present in all kingdoms of life and their specific roles remain enigmatic. More studies are needed to unravel the crucial functions of those interesting proteins.


Assuntos
Coenzimas/química , Metais/química , Proteínas Quinases/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Domínio Catalítico , Cátions Bivalentes/química , Cátions Bivalentes/metabolismo , Coenzimas/metabolismo , Complexos de Coordenação , Eucariotos/metabolismo , Evolução Molecular , Humanos , Metais/metabolismo , Proteínas Quinases/química , Proteínas Quinases/genética
11.
N Biotechnol ; 33(5 Pt A): 574-81, 2016 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26709003

RESUMO

Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Cadeia Única , Especificidade de Anticorpos , Biotecnologia , Humanos , Imuno-Histoquímica , Projetos Piloto , Análise Serial de Proteínas , Controle de Qualidade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética
12.
ACS Chem Biol ; 10(10): 2303-15, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26200257

RESUMO

cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca(2+), do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination properties. We found that Ca(2+), in contrast to Mg(2+), does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca(2+) works in a single turnover mode to allow for phosphoryl-transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with a substrate in the presence of Mg(2+)ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg(2+), Ca(2+) apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca(2+) is not as efficient as Mg(2+) in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg(2+) easily releases the phospho-substrate while Ca(2+) traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca(2+). Furthermore, Ca(2+) is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg(2+). We therefore hypothesize that the physiological metal ions Mg(2+) and Ca(2+) both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca(2+), our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling.


Assuntos
Cálcio/farmacologia , Cátions Bivalentes/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Magnésio/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/química , Cátions Bivalentes/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Ativação Enzimática/efeitos dos fármacos , Íons , Magnésio/química , Modelos Biológicos , Dados de Sequência Molecular , Alinhamento de Sequência
13.
PLoS Biol ; 13(7): e1002192, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26158466

RESUMO

To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RIIß holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIß subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIß holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIß holoenzyme since the RIIß holoenzyme is located close to ion channels. The 2.8Å structure of an RIIßp2:C2:(Ca2ADP)2 holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(ß,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a "product trap" because of the slow off-rate of the pRIIß subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIß signaling cycle, we show that release of pRIIß in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIß holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes.


Assuntos
Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Catálise , AMP Cíclico/metabolismo , Escherichia coli , Células HeLa , Holoenzimas/metabolismo , Humanos , Magnésio/metabolismo , Camundongos , Células NIH 3T3 , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Ratos
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