RESUMO
Proteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions.
Assuntos
Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Humanos , Fosfatos de Inositol/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestrutura , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Ligação Proteica , Conformação Proteica , Proteólise , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera , Relação Estrutura-AtividadeRESUMO
During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function, and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82 to 92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners and dictate its role in germ cell differentiation.
Assuntos
Complexo de Endopeptidases do Proteassoma , Espermatogênese , Espermatogônias , Humanos , Masculino , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Espermatogônias/enzimologiaRESUMO
The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the ß2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum ß2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this ß2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Desenho de Fármacos , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Animais , Antimaláricos/efeitos adversos , Antimaláricos/toxicidade , Artemisininas/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Sinergismo Farmacológico , Ativação Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Plasmodium/crescimento & desenvolvimento , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium chabaudi/enzimologia , Plasmodium chabaudi/fisiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Inibidores de Proteassoma/efeitos adversos , Inibidores de Proteassoma/toxicidade , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade da Espécie , Especificidade por Substrato/efeitos dos fármacosRESUMO
Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the ß2, ß5 or ß6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas de Protozoários , Antimaláricos/química , Resistência a Medicamentos/genética , Sinergismo Farmacológico , Humanos , Plasmodium falciparum/genética , Inibidores de Proteassoma/química , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The 26S proteasome plays a fundamental role in eukaryotic homeostasis by undertaking the highly controlled degradation of a wide range of proteins, including key cellular regulators such as those controlling cell-cycle progression and apoptosis. Here we report the structure of the human 26S proteasome determined by cryo-electron microscopy and single-particle analysis, with secondary structure elements identified both in the 20S proteolytic core region and in the 19S regulatory particle. We have used this information together with crystal structures, homology models, and other biochemical information to construct a molecular model of the complete 26S proteasome. This model allows for a detailed description of the 20S core within the 26S proteasome and redefines the overall assignment of subunits within the 19S regulatory particle. The information presented here provides a strong basis for a mechanistic understanding of the 26S proteasome.
Assuntos
Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/química , Humanos , Estrutura Quaternária de ProteínaRESUMO
The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.
Assuntos
Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase , Biocatálise , Linhagem Celular , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestrutura , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Peso Molecular , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Espalhamento de Radiação , Schizosaccharomyces/química , Relação Estrutura-Atividade , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/ultraestrutura , UbiquitinaçãoRESUMO
The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at â¼10 Å resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase , Biocatálise , Proteínas Cdh1 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/ultraestrutura , UbiquitinaçãoRESUMO
Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas Recombinantes/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc7 do Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP(3)R) channel function. The residues selected were highly conserved in all three IP(3)R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate. Of five negatively charged residues mutated, none completely eliminated channel function. Of five positively charged residues mutated, only one inactivated the channel (Arg-2596). In addition to the previously identified role of a pair of cysteines in the C-tail (Cys-2610 and Cys-2613), a pair of highly conserved histidines (His-2630 and His-2635) were also essential for channel function. Expression of the H2630A and H2635A mutants (but not R2596A) produced receptors with destabilized interactions between the N-terminal fragment and the channel domain. A previously unrecognized association between the cytosolic C-tail and the TM 4,5-loop was demonstrated using GST pulldown assays. However, none of the mutations in the C-tail interfered with this interaction or altered the ability of the C-tail to assemble into dimers. Our present findings and recent information on IP(3)R structure from electron microscopy and crystallography are incorporated into a revised model of channel gating.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato , Ativação do Canal Iônico/fisiologia , Modelos Moleculares , Multimerização Proteica/fisiologia , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RatosRESUMO
The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14-15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Cristalografia por Raios X , Humanos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Homologia de Sequência de AminoácidosRESUMO
The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break.
Assuntos
Proteína Quinase Ativada por DNA/ultraestrutura , DNA/metabolismo , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Dimerização , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica/métodos , FosforilaçãoRESUMO
The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic ß2 and ß5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the ß5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.
Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Plasmodium , Humanos , Antimaláricos/farmacologia , Antimaláricos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Plasmodium/metabolismo , Artemisininas/química , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/químicaRESUMO
Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Apresentação de Antígeno , Neoplasias Pulmonares/patologia , Medicina de Precisão , Complexo de Endopeptidases do Proteassoma/metabolismo , Microambiente TumoralRESUMO
We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca(2+)-induced conformational changes of IP(3)Rs in their native membrane environment. We found that Ca(2+) decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca(2+) induces a conformational change in the IP(3)R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca(2+) were observed at approximately 0.5 microm and was sensitive to inhibition by IP(3). Ca(2+) also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1-225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca(2+). However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca(2+). We conclude the following: a) that large conformational changes induced by Ca(2+) can be detected in IP(3)Rs in situ; b) these changes may be driven by Ca(2+) binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP(3)R may be relatively inaccessible to regulatory proteins unless Ca(2+) is present.
Assuntos
Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/química , Animais , Sítios de Ligação , Células COS , Cálcio/química , Chlorocebus aethiops , Cisteína/química , Epitopos/química , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Mutação , Fosforilação , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Inositol (1,4,5)-trisphosphate receptors (IP(3)R) are intracellular Ca(2+) channels that are regulated by Ca(2+) and IP(3), and are modulated by many additional signals. They thereby allow both receptors that stimulate IP(3) formation and Ca(2+) to control release of Ca(2+) from intracellular stores. IP(3)Rs share many features with their close relatives, ryanodine receptors; each provides insight into the structure and function of the other. The structural basis of IP(3)R behaviour is beginning to emerge from intermediate-resolution structures of the complete IP(3)R, a 2.2-A structure of the IP(3)-binding core and comparisons with the pore structures of other tetrameric cation channels. The binding of IP(3) to a site towards the N-terminal of each IP(3)R subunit promotes binding of Ca(2+). This destabilizes an inhibitory interaction between N-terminal residues and a C-terminal 'gatekeeper' sequence, enabling the pore to open.
Assuntos
Canais de Cálcio/química , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Homologia de Sequência de AminoácidosRESUMO
With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein-ligand interactions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein-ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites. Subsequently, the cryo-EM structure of the Plasmodium falciparum 20S proteasome, with a new prototype specific inhibitor bound, revealed the molecular basis for the ligand specificity towards the parasite complex, which provides a framework to guide the development of highly needed new-generation antimalarials. Here, the cryo-EM analysis of the ligand-bound human and P. falciparum 20S proteasomes is reviewed, and a complete description of the methods used for structure determination is provided, including the strategy to overcome the bias orientation of the human 20S proteasome on electron-microscope grids and details of the icr3d software used for three-dimensional reconstruction.
Assuntos
Antimaláricos/farmacologia , Microscopia Crioeletrônica/métodos , Plasmodium falciparum/enzimologia , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Inibidores de Proteassoma/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Plasmodium falciparum/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Plasmodium falciparum is the parasite responsible for the most severe form of malaria. Its increasing resistance to existing antimalarials represents a major threat to human health and urges the development of new therapeutic strategies to fight malaria. The proteasome is a protease complex essential in all eukaryotes. Accordingly, inhibition of the Plasmodium 20S proteasome is highly toxic for the parasite at all of its infective and developmental stages. Proteasome inhibitors have antimalarial potential both as curative and transmission blocking agents, but in order to have therapeutic application, they must specifically target the Plasmodium proteasome and not its human counterpart. X-ray crystallography has been widely used to determine structures of yeast and mammalian 20S proteasomes with ligands. However, crystallisation of the Plasmodium proteasome is challenging, as only small quantities of the complex can be directly purified from the parasite. Furthermore, most X-ray structures of proteasome-inhibitor complexes require soaking of crystals with high concentrations of ligand, thus preventing analysis of inhibitor subunit specificity. Instead we chose to determine the Plasmodium falciparum 20S proteasome structure, in the presence of a new rationally designed parasite-specific inhibitor, by high-resolution electron cryo-microscopy and single particle analysis. The resulting map, at a resolution of about 3.6 Å, allows a direct molecular analysis of inhibitor/enzyme interactions. Here we present an overview of this structure, and how it provides valuable information that can be used to assist in the design of improved proteasome inhibitors with the potential to be developed as next-generation antimalarial drugs.
Assuntos
Microscopia Crioeletrônica/métodos , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Inibidores de Proteassoma/farmacologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Malária Falciparum/parasitologia , Modelos Moleculares , Plasmodium falciparum/fisiologia , Plasmodium falciparum/ultraestrutura , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression. It functions by removing a wide range of specifically tagged proteins, including key cellular regulators. Here we present the structure of the human 20S proteasome core bound to a substrate analogue inhibitor molecule, determined by electron cryo-microscopy (cryo-EM) and single-particle analysis at a resolution of around 3.5 Å. Our map allows the building of protein coordinates as well as defining the location and conformation of the inhibitor at the different active sites. These results open new prospects to tackle the proteasome functional mechanisms. Moreover, they also further demonstrate that cryo-EM is emerging as a realistic approach for general structural studies of protein-ligand interactions.