RESUMO
The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Šresolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.
Assuntos
Artemisininas/farmacologia , Regulação para Baixo , Inibição Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridoxal Quinase/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Artemisininas/química , Sítios de Ligação , Regulação para Baixo/efeitos dos fármacos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Piridoxal Quinase/química , Piridoxal Quinase/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Ácido gama-Aminobutírico/biossínteseRESUMO
Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide-clearance cascade. Inâ vitro and inâ vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra- and intermolecular inhibitor-inhibitor, protein-protein, and inhibitor-protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.
Assuntos
Antiprotozoários/química , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Oxirredutases/química , Tiorredoxinas/química , Trypanosoma brucei brucei/enzimologia , Animais , Antiprotozoários/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Glutationa/análogos & derivados , Glutationa/química , Humanos , Peróxido de Hidrogênio/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Espermidina/análogos & derivados , Espermidina/química , Trypanosoma/metabolismo , Trypanosoma/parasitologiaRESUMO
The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.
Assuntos
Regulação Alostérica , Proteínas de Transporte/análise , Proteínas de Membrana/análise , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sinapses/química , Sinapses/fisiologia , Animais , Membrana Celular/química , Células Cultivadas , Cristalografia por Raios X , Camundongos , Microscopia de Força Atômica , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Espalhamento a Baixo ÂnguloRESUMO
The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.
RESUMO
Targeting multiprotein receptor complexes, rather than receptors directly, is a promising concept in drug discovery. This is particularly relevant to the GABAB receptor complex, which plays a prominent role in many brain functions and diseases. Here, we provide the first studies targeting a key protein-protein interaction of the GABAB receptor complex-the interaction with KCTD proteins. By employing the µSPOT technology, we first defined the GABAB receptor-binding epitope mediating the KCTD interaction. Subsequently, we developed a highly potent peptide-based inhibitor that interferes with the KCTD/GABAB receptor complex and efficiently isolates endogenous KCTD proteins from mouse brain lysates. X-ray crystallography and SEC-MALS revealed inhibitor induced oligomerization of KCTD16 into a distinct hexameric structure. Thus, we provide a template for modulating the GABAB receptor complex, revealing a fundamentally novel approach for targeting GABAB receptor-associated neuropsychiatric disorders.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Receptores de GABA-B/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/metabolismo , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de GABA-B/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
TRPML2 is the least structurally characterized mammalian transient receptor potential mucolipin ion channel. The TRPML family hallmark is a large extracytosolic/lumenal domain (ELD) between transmembrane helices S1 and S2. We present crystal structures of the tetrameric human TRPML2 ELD at pH 6.5 (2.0 Å) and 4.5 (2.95 Å), corresponding to the pH values in recycling endosomes and lysosomes. Isothermal titration calorimetry shows Ca2+ binding to the highly acidic central pre-pore loop which is abrogated at low pH, in line with a pH-dependent channel regulation model. Small angle X-ray scattering confirms the ELD dimensions in solution. Changes in pH or Ca2+ concentration do not affect the protein's secondary structure, but can influence ELD oligomer integrity according to native mass spectrometry. Our data thus complete the set of high-resolution views of human TRPML channel ELDs and reveal some structural responses to the conditions the TRPML2 ELD encounters as the channel traffics through the endolysosomal system.
Assuntos
Cálcio/metabolismo , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Glioblastomas (GBMs) are the most frequent malignant brain tumors with very limited treatment options and nearly all GBM patients dying within 1 year. Pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) is a secreted growth factor that shows mitogenic, chemotactic and transforming activity. While PTN expression is tightly regulated during embryogenesis and very limited in normal adult tissues, a marked PTN upregulation is seen in tumors including glioblastomas. Targeting of the PTN receptors, ALK and RPTP-zeta, indicates a contribution of PTN-activated signaling pathways in glioblastomas. However, the relevance of PTN expression itself is unknown especially since, besides PTN, at least one more growth factor, midkine (MK), signals through ALK and is expressed in glioblastoma. Here we demonstrate the biologic relevance of PTN in 2 glioblastoma cell lines in vitro and in vivo. We show that stable ribozyme-targeting leads to a robust reduction of PTN mRNA and protein levels. This results in decreased cell proliferation, cell migration and soft agar colony formation in vitro. Comparing clonal ribozyme-transfected cells with different residual PTN levels, we establish a PTN gene-dose effect of glioblastoma cell proliferation. In a subcutaneous tumor xenograft mouse model, in vivo growth is markedly reduced upon PTN depletion, which is paralleled by decreased PTN serum levels. Furthermore, the immunohistochemical analysis of the tumors shows reduced angiogenesis in PTN-depleted tumors. We conclude that PTN is a rate-limiting growth factor in glioblastoma. Since PTN is overexpressed in glioblastomas but rarely found in normal tissue, PTN may represent an attractive therapeutic target.