RESUMO
C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.
Assuntos
Retículo Endoplasmático , Humanos , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/química , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/química , Ligação ProteicaRESUMO
Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.
Assuntos
Espectrometria de Massas em Tandem , Raios Ultravioleta , Humanos , Espectrometria de Massas em Tandem/métodos , Cisteína/química , Cisteína/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligação Proteica , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Modelos MolecularesRESUMO
A normal phosphorylation state is essential for the function of proteins. Biased regulation frequently results in morbidity, especially for the hyperphosphorylation of oncoproteins. The hyperphosphorylation of ASK1 at Thr838 leads to a persistently high activity state, which accelerates the course of gastric cancer. Under normal conditions, PP5 specifically dephosphorylates p-ASK1T838 in cells, thereby weakening ASK1 to a low-basal activity state. However, in tumor types, PP5 shows low activity with a self-inhibition mechanism, making p-ASK1T838 remain at a high level. Thus, we aim to design phosphatase recruitment chimeras (PHORCs) through a proximity-mediated effect for specifically accelerating the dephosphorylation of p-ASK1T838. Herein, we describe DDO3711 as the first PP5-recruiting PHORC, which is formed by connecting a small molecular ASK1 inhibitor to a PP5 activator through a chemical linker, to effectively decrease the level of p-ASK1T838 in vitro and in vivo. DDO3711 shows preferable antiproliferative activity (IC50 = 0.5 µM) against MKN45 cells through a direct binding and proximity-mediated mechanism, while the ASK1 inhibitor and the PP5 activator, used alone or in combination, exhibit no effect on MKN45 cells. Using DDO3711, PHORCs are identified as effective tools to accelerate the dephosphorylation of POIs and provide important evidence to achieve precise phosphorylation regulation, which will promote confidence in the further regulation of abnormally phosphorylated oncoproteins.
Assuntos
MAP Quinase Quinase Quinase 5 , Fosfoproteínas Fosfatases , Apoptose , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Transdução de Sinais , Antineoplásicos/química , MAP Quinase Quinase Quinase 5/químicaRESUMO
Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.
Assuntos
Fosfoproteínas Fosfatases , Proteômica , Espectrometria de Massas , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteômica/métodosRESUMO
CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+ and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.
Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Fosfoproteínas Fosfatases , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/química , Oxirredução , Fosfoproteínas Fosfatases/química , Fosforilação , Carbonilação Proteica , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismoRESUMO
PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Estresse Fisiológico , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Ativação Enzimática , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Oxirredução , Fosfoproteínas Fosfatases/química , Fosforilação , Ligação Proteica , Domínios Proteicos , RNA Interferente Pequeno/metabolismoRESUMO
Over the last decades, research has focused on the role of pleckstrin homology (PH) domain leucine-rich repeat protein phosphatases (PHLPPs) in regulating cellular signaling via PI3K/Akt inhibition. The PKB/Akt signaling imbalances are associated with a variety of illnesses, including various types of cancer, inflammatory response, insulin resistance, and diabetes, demonstrating the relevance of PHLPPs in the prevention of diseases. Furthermore, identification of novel substrates of PHLPPs unveils their role as a critical mediator in various cellular processes. Recently, researchers have explored the increasing complexity of signaling networks involving PHLPPs whereby relevant information of PHLPPs in metabolic diseases was obtained. In this review, we discuss the current knowledge of PHLPPs on the well-known substrates and metabolic regulation, especially in liver, pancreatic beta cell, adipose tissue, and skeletal muscle in relation with the stated diseases. Understanding the context-dependent functions of PHLPPs can lead to a promising treatment strategy for several kinds of metabolic diseases. [BMB Reports 2021; 54(9): 451-457].
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Tecido Adiposo/metabolismo , Humanos , Resistência à Insulina , Fígado/metabolismo , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Especificidade por SubstratoRESUMO
Phosphoprotein phosphatase (PPP) enzymes are ubiquitous proteins involved in cellular signaling pathways and other functions. Here we have traced the origin of the PPP sequences of Eukaryotes and their radiation. Using a bacterial PPP Hidden Markov Model (HMM) we uncovered "BacterialPPP-Like" sequences in Archaea. A HMM derived from eukaryotic PPP enzymes revealed additional, unique sequences in Archaea and Bacteria that were more like the eukaryotic PPP enzymes then the bacterial PPPs. These sequences formed the basis of phylogenetic tree inference and sequence structural analysis allowing the history of these sequence types to be elucidated. Our phylogenetic tree data strongly suggest that eukaryotic PPPs ultimately arose from ancestors in the Asgard archaea. We have clarified the radiation of PPPs within Eukaryotes, substantially expanding the range of known organisms with PPP subtypes (Bsu1, PP7, PPEF/RdgC) previously thought to have a more restricted distribution. Surprisingly, sequences from the Methanosarcinaceae (Euryarchaeota) form a strongly supported sister group to eukaryotic PPPs in our phylogenetic analysis. This strongly suggests an intimate association between an Asgard ancestor and that of the Methanosarcinaceae. This is highly reminiscent of the syntrophic association recently demonstrated between the cultured Lokiarchaeal species Prometheoarchaeum and a methanogenic bacterial species.
Assuntos
Archaea/enzimologia , Bactérias/enzimologia , Eucariotos/enzimologia , Fosfoproteínas Fosfatases/química , Sequência de Aminoácidos , Animais , Archaea/química , Archaea/genética , Bactérias/química , Bactérias/genética , Eucariotos/química , Eucariotos/genética , Evolução Molecular , Humanos , Fosfoproteínas Fosfatases/genética , FilogeniaRESUMO
Protein phosphorylation is a major reversible post-translational modification. Protein phosphatases function as 'critical regulators' in signaling networks through dephosphorylation of proteins, which have been phosphorylated by protein kinases. A large understanding of their working has been sourced from animal systems rather than the plant or the prokaryotic systems. The eukaryotic protein phosphatases include phosphoprotein phosphatases (PPP), metallo-dependent protein phosphatases (PPM), protein tyrosine (Tyr) phosphatases (PTP), and aspartate (Asp)-dependent phosphatases. The PPP and PPM families are serine(Ser)/threonine(Thr)-specific phosphatases (STPs), while PTP family is Tyr specific. Dual-specificity phosphatases (DsPTPs/DSPs) dephosphorylate Ser, Thr, and Tyr residues. PTPs lack sequence homology with STPs, indicating a difference in catalytic mechanisms, while the PPP and PPM families share a similar structural fold indicating a common catalytic mechanism. The catalytic cysteine (Cys) residue in the conserved HCX5 R active site motif of the PTPs acts as a nucleophile during hydrolysis. The PPP members require metal ions, which coordinate the phosphate group of the substrate, followed by a nucleophilic attack by a water molecule and hydrolysis. The variable holoenzyme assembly of protein phosphatase(s) and the overlap with other post-translational modifications like acetylation and ubiquitination add to their complexity. Though their functional characterization is extensively reported in plants, the mechanistic nature of their action is still being explored by researchers. In this review, we exclusively overview the plant protein phosphatases with an emphasis on their mechanistic action as well as structural characteristics.
Assuntos
Domínio Catalítico , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Plantas/metabolismo , Domínios Proteicos , Transdução de Sinais , Biocatálise , Modelos Moleculares , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/classificação , Fosforilação , Proteínas de Plantas/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por SubstratoRESUMO
The spermatozoa are transcriptionally dormant entities which have been recognized to be an archive of mRNA, coding for a variety of functionally crucial cellular proteins. This significant repository of mRNA is predicted to be associated with early embryogenesis and postfertilization. The mRNA transcripts which are tagged with minisatellites have been involved in the regulation of the gene functions as well as their organization. However, very little information is available regarding the expression of the transcripts tagged with minisatellites in spermatozoa. Therefore, in order to understand the functions and the conformational behavior of the proteins expressed from these minisatellite-tagged transcripts, we have performed a detailed in silico analysis using the sequences of the transcripts. The protein predicted from KF274549 showed the functionalities similar to uncharacterized C4orf26 proteins, while that obtained from KF274557 predicted to be a metallophosphoesterase. Furthermore, the structural folds in the structure of these predicted proteins were analyzed by using the homology modeling and their conformational behaviors in the explicit water conditions were analyzed by using the techniques of Molecular Dynamics (MD) simulations. This detailed analysis will facilitate the understanding of these proteins in the spermatozoon region and can be used for uncovering other attributes of the metabolic network.
Assuntos
Repetições Minissatélites/genética , Fosfoproteínas Fosfatases , Proteínas de Transporte/química , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteínas Nucleares/química , Proteínas Nucleares/classificação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Conformação Proteica , Proteínas Proto-Oncogênicas c-aktRESUMO
Protein phosphatase 5 (PP5) is a serine/threonine protein phosphatase that regulates many cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is also a co-chaperone of the heat shock protein 90 molecular chaperone machinery that assists in regulation of cellular signaling pathways essential for cell survival and growth. PP5 plays a significant role in survival and propagation of multiple cancers, which makes it a promising target for cancer therapy. Though there are several naturally occurring PP5 inhibitors, none is specific for PP5. Here, we review the roles of PP5 in cancer progression and survival and discuss the unique features of the PP5 structure that differentiate it from other phosphoprotein phosphatase (PPP) family members and make it an attractive therapeutic target.
Assuntos
Neoplasias/enzimologia , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/fisiologia , Neoplasias da Mama/enzimologia , Domínio Catalítico , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismoRESUMO
The PHLPP (Pleckstrin homology domain leucine-rich repeat protein phosphatases) is a newly discovered group of genes which includes PHLPP1 and PHLPP2 and plays an integral part in several cellular processes like apoptosis, cell signaling cell survival, and cell proliferation etc. Both the activation and deactivation of these genes can have vital role in several ailments like heart diseases, circadian rhythm and most importantly the cancer, hence encouraging the growth of novel therapeutic elements. To give new directions into the development of PHLPP1- targeting drugs, the interaction mechanism between PHLPP1 and five important ligands 4IP, B39, 635, ATP and GTA were investigated through docking and Molecular Dynamics Simulation. It is also noteworthy to be mentioned here that there is no previous crystal structure of PHLPP1 available. The in-silico results can provide potential base for advancements in development of new therapeutic elements targeting different diseases, mainly cancer. In this study, we employed homology modeling technique to develop a high-quality structure model of PHLPP1. The PHLPP1 model was then used in docking interaction analysis and Molecular Dynamics Simulation, to study binding pockets and interactions of PHLPP1 ligands and finding actively contributing residues in binding pocket. In final step, Free Energy Estimation was performed to observe ligand binding's quantitative characteristics.
Assuntos
Simulação por Computador , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Humanos , Proteínas Nucleares/química , Fosfoproteínas Fosfatases/química , Ligação Proteica/fisiologiaRESUMO
Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.
Assuntos
Aurora Quinase A/genética , Caseína Quinase II/química , Fosfoproteínas Fosfatases/genética , Alanina/genética , Substituição de Aminoácidos/genética , Aurora Quinase A/química , Sistemas CRISPR-Cas/genética , Caseína Quinase II/genética , Domínio Catalítico/genética , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosforilação/genética , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Especificidade por Substrato/efeitos dos fármacosRESUMO
Protein-protein interaction network analysis plays critical roles in predicting the functions of target proteins. In this study, we used a combination of SILAC-MS proteomics and bioinformatic approaches to identify Checkpoint Kinase 1 (Chk1) as a possible POPX2 phosphatase interacting protein. POPX2 is a PP2C phosphatase that has been implicated in cancer cell invasion and migration. From the Domain-Domain Interaction (DDI) database, we first determined that the PP2C phosphatase domain interacts with Pkinase domain. Subsequently, 46 proteins with Pkinase domain were identified from POPX2 SILAC-MS data. We then narrowed down the leads and confirmed the biological interaction between Chk1 and POPX2. We also found that Chk1 is a substrate of POPX2. Chk1 is a key regulator of the cell cycle and is activated when the cell suffers DNA damage. Our approach has led us to identify POPX2 as a regulator of Chk1 and can interfere with the normal function of Chk1 at G1-S transition of the cell cycle in response to DNA damage.
Assuntos
Ciclo Celular , Quinase 1 do Ponto de Checagem/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Dano ao DNA , Inativação Gênica , Humanos , Modelos Biológicos , Fosfoproteínas Fosfatases/química , Fosforilação , Filogenia , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Reprodutibilidade dos Testes , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Protein phosphatase 5 (PP5), an important member of serine/threonine protein phosphatases, has been associated with diverse human cancers. Structural data demonstrate that two metal ions and two water molecules are located in the active site of PP5 and are involved in the dephosphorylations of the substrate. Recently, the catalytic reaction of PP5 has been studied by a density functional theory (DFT) method, and the role of the water molecules in regulating the substrate binding of PP5 was explored by our previous molecular dynamics (MD) study. These two studies enrich our understanding of the catalytic mechanism of PP5. However, the questions of how the metal ions regulate the substrate binding and the contribution of each metal ion to the catalysis remain not answered. To this end, the role of the catalytic Mn2+ ions in regulating the binding of PP5 with its substrate was investigated through MD simulations. Results reveal that the different states of Mn2+ ions can alter the structure of the active site and the conformations of the residues coordinating the Mn2+ ions or the substrate. This leads to changes in the secondary structure and dynamical fluctuation of PP5, especially for that of helix αJ. With the position change of helix αJ, the volume of the substrate binding pocket is altered by the different states of Mn2+ ions, indicating the regulatory role of Mn2+ ions in PP5 activity. Furthermore, combined with the simulation data of our previous study, the sequential steps for PP5 binding of the substrate, the two active site water molecules, and the two Mn2+ ions are proposed. This may help to develop new inhibitors to treat the PP5-related cancers.
Assuntos
Magnésio/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sítios de Ligação , Domínio Catalítico , Cátions Bivalentes/metabolismo , Humanos , Magnésio/química , Simulação de Dinâmica Molecular , Proteínas Nucleares/química , Fosfoproteínas Fosfatases/química , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
In bull spermatozoa, extracellular Ca2+-dependent full-type hyperactivation, which is characterized by the asymmetrical beating in whole parts of the middle/principal pieces, is suppressed by calyculin A-sensitive protein phosphatases. The aim of this study was to identify isoforms of these protein phosphatases. Ejaculated spermatozoa were used for the investigation on effects of protein phosphatase inhibitors (calyculin A with high specificity for both of protein phosphatases 1 and 2A, and okadaic acid with relatively higher specificity for protein phosphatase 2A than protein phosphatase 1) on the induction of extracellular Ca2+-dependent full-type hyperactivation by incubation with CaCl2 and cAMP analog (cBiMPS). They were also used for the immunodetection of protein phosphatases 1α, 1ß, 1γ, 2Aα and 2Aß. Percentages of full-type hyperactivated spermatozoa significantly increased after incubation with calyculin A (10â¯nM) in a concentration-dependent manner of CaCl2 (0-3.42â¯mM), though only minor increases in the percentages of full-type hyperactivated spermatozoa were observed after incubation with okadaic acid (10â¯nM). Moreover, the immunodetection of protein phosphatase isoforms showed sperm connecting piece and flagellum included protein phosphatases 1α and 1γ, but did not do the other isoforms. These results suggest that calyculin A-sensitive and okadaic acid-less sensitive protein phosphatases (1α and 1γ) are suppressors for the extracellular Ca2+-dependent full-type hyperactivation in bull ejaculated spermatozoa.
Assuntos
Bovinos , Fosfoproteínas Fosfatases/fisiologia , Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Toxinas Marinhas , Oxazóis , Fosfoproteínas Fosfatases/química , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
LB-100 is an experimental cancer therapeutic with cytotoxic activity against cancer cells in culture and antitumor activity in animals. The first phase I trial (NCT01837667) evaluating LB-100 recently concluded that safety and efficacy parameters are favorable for further clinical testing. Although LB-100 is widely reported as a specific inhibitor of serine/threonine phosphatase 2A (PP2AC/PPP2CA:PPP2CB), we could find no experimental evidence in the published literature demonstrating the specific engagement of LB-100 with PP2A in vitro, in cultured cells, or in animals. Rather, the premise for LB-100 targeting PP2AC is derived from studies that measure phosphate released from a phosphopeptide (K-R-pT-I-R-R) or inferred from the ability of LB-100 to mimic activity previously reported to result from the inhibition of PP2AC by other means. PP2AC and PPP5C share a common catalytic mechanism. Here, we demonstrate that the phosphopeptide used to ascribe LB-100 specificity for PP2A is also a substrate for PPP5C. Inhibition assays using purified enzymes demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C. The structure of PPP5C cocrystallized with LB-100 was solved to a resolution of 1.65Å, revealing that the 7-oxabicyclo[2.2.1]heptane-2,3-dicarbonyl moiety coordinates with the metal ions and key residues that are conserved in both PP2AC and PPP5C. Cell-based studies revealed some known actions of LB-100 are mimicked by the genetic disruption of PPP5C These data demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C and suggest that the observed antitumor activity might be due to an additive effect achieved by suppressing both PP2A and PPP5C.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Neoplasias/tratamento farmacológico , Proteínas Nucleares/química , Fosfoproteínas Fosfatases/química , Piperazinas/química , Proteína Fosfatase 2/química , Sequência de Aminoácidos/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Catálise , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Metais/química , Metilação , Mutagênese Sítio-Dirigida , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/antagonistas & inibidores , Fosfoproteínas Fosfatases/antagonistas & inibidores , Piperazinas/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genéticaRESUMO
According to the sequence and profile comparison with known catalytic domains, where identified protein phosphatases potentially involved in regulation of microtubule dynamics and structure from Arabidopsis thaliana, Nicotiana tabacum, Medicago sativa, Oryza sativa subsp. japonica, Zea mays, and Triticum aestivum. Selected proteins were related to classical non-receptor, serine/threonine-specific and dual protein phosphatases. By application of template structures of human protein phosphatases, it was performed homology modelling of the catalytic domains of 17 plant protein phosphatases. Based on the results of the structural alignment, molecular dynamics, and conservatism in positions of functionally importance, it was confirmed homology of selected plant proteins and known protein phosphatases regulating structure and dynamics of microtubules.
Assuntos
Microtúbulos/metabolismo , Fosfoproteínas Fosfatases/química , Proteínas de Plantas/química , Plantas/enzimologia , Domínio Catalítico , Humanos , Fosfoproteínas Fosfatases/genética , Proteínas de Plantas/genética , Homologia Estrutural de ProteínaRESUMO
Mammalian haloacid dehalogenase (HAD)-type phosphatases have evolved to dephosphorylate a wide range of small metabolites, but can also target macromolecules such as serine/threonine, tyrosine-, and histidine-phosphorylated proteins. To accomplish these tasks, HAD phosphatases are equipped with cap domains that control access to the active site and provide substrate specificity determinants. A number of capped HAD phosphatases impact protein phosphorylation, although structural data are consistent with small metabolite substrates rather than protein substrates. This review discusses the structures, functions and disease implications of the three closely related, capped HAD phosphatases pyridoxal phosphatase (PDXP or chronophin), phosphoglycolate phosphatase (PGP, also termed AUM or glycerol phosphatase) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP or HDHD2B). Evidence in support of small metabolite and protein phosphatase activity is discussed in the context of the diversity of their biological functions.
Assuntos
Pirofosfatase Inorgânica/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Humanos , Hidrolases , Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/fisiologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/fisiologia , Fosfoproteínas Fosfatases/ultraestrutura , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/fisiologia , Fosforilação , Proteínas Tirosina Fosfatases/metabolismoRESUMO
The serine/threonine protein phosphatase type 5 (PP5) is a promising target for designing new antitumor drugs. This enzyme is a member of the PPP phosphatases gene family, which catalyzes a dephosphorylation reaction: a regulatory process in the signal transduction pathway that controls various biological processes. The aim of this work is to study and compare the inhibition of PP5 by ten cantharidin-like inhibitors in order to bring about contributions relevant to the better comprehension of their inhibitory activity. In this theoretical investigation, we used molecular dynamics techniques to understand the role of key interactions that occur in the protein active site; QM calculations were employed to study the interaction mode of these inhibitors in the enzyme. In addition, atoms in molecules (AIM) calculations were carried out to characterize the chemical bonds among the atoms involved and investigate the orbital interactions with their respective energy values. The obtained results suggest that the Arg275, Asn303, His304, His352, Arg400, His427, Glu428, Val429, Tyr451, and Phe446 residues favorably contribute to the interactions between inhibitors and PP5. However, the Asp271 and Asp244 amino acid residues do not favor such interactions for some inhibitors. Through the QM calculations, we can suggest that the reactional energy of the coordination mechanism of these inhibitors in the PP5 active site is quite important and is responsible for the inhibitory activity. The AIM technique employed in this work was essential to get a better comprehension of the transition states acquired from the mechanism simulation. This work offers insights of how cantharidin-like inhibitors interact with human PP5, potentially allowing the design of more specific and even less cytotoxic drugs for cancer treatments. Graphical Abstract Interactions of cantharidin-like inhibitors with human protein phosphatase-5 in a Mg2+ system.