RESUMO
Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.
Assuntos
Sítio Alostérico , Antineoplásicos/farmacologia , Proteínas de Choque Térmico/antagonistas & inibidores , Serina Peptidase 1 de Requerimento de Alta Temperatura A/antagonistas & inibidores , Proteínas Periplásmicas/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Regulação Alostérica , Antineoplásicos/química , Escherichia coli , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/química , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Proteínas Periplásmicas/química , Proteínas Periplásmicas/metabolismo , Inibidores de Proteases/química , Ligação Proteica , Serina Endopeptidases/química , Serina Endopeptidases/metabolismoRESUMO
Protein quality control involves sensing and treatment of defective or incomplete protein structures. Misfolded or mislocalized proteins trigger dedicated signal transduction cascades that upregulate the production of protein quality-control factors. Corresponding proteases and chaperones either degrade or repair damaged proteins, thereby reducing the level of aggregation-prone molecules. Because the periplasm of gram-negative bacteria is particularly exposed to environmental changes and respective protein-folding stresses connected with the presence of detergents, low or high osmolarity of the medium, elevated temperatures, and the host's immune response, fine-tuned protein quality control systems are essential for survival under these unfavorable conditions. This review discusses recent advances in the identification and characterization of the key cellular factors and the emerging general principles of the underlying molecular mechanisms.
Assuntos
Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Animais , Bactérias/química , Bactérias/genética , Infecções Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Humanos , Periplasma/química , Periplasma/genética , Dobramento de Proteína , Transporte ProteicoRESUMO
The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydiaâ CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.
Assuntos
Antibacterianos/metabolismo , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/enzimologia , Dipeptídeos/metabolismo , Corpos de Inclusão/microbiologia , Viabilidade Microbiana/efeitos dos fármacos , Organofosfonatos/metabolismo , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/metabolismo , Linhagem Celular , Chlamydia trachomatis/genética , Genes Essenciais , Hepatócitos/microbiologia , Humanos , MicroscopiaRESUMO
Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.
Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Animais , Humanos , Camundongos , Conformação Proteica , Multimerização Proteica , Células HEK293 , Encéfalo/metabolismo , Encéfalo/patologia , Mutação , Mutação com Perda de FunçãoRESUMO
The efficient intracellular delivery of (bio)molecules into living cells remains a challenge in biomedicine. Many biomolecules and synthetic drugs are not able to cross the cell membrane, which is a problem if an intracellular mode of action is desired, for example, with a nuclear receptor. Calcium phosphate nanoparticles can serve as carriers for small and large biomolecules as well as for synthetic compounds. The nanoparticles were prepared and colloidally stabilized with either polyethyleneimine (PEI; cationic nanoparticles) or carboxymethyl cellulose (CMC; anionic nanoparticles) and loaded with defined amounts of the fluorescently labelled proteins HTRA1, HTRA2, and BSA. The nanoparticles were purified by ultracentrifugation and characterized by dynamic light scattering and scanning electron microscopy. Various cell types (HeLa, MG-63, THP-1, and hMSC) were incubated with fluorescently labelled proteins alone or with protein-loaded cationic and anionic nanoparticles. The cellular uptake was followed by light and fluorescence microscopy, confocal laser scanning microscopy (CLSM), and flow cytometry. All proteins were readily transported into the cells by cationic calcium phosphate nanoparticles. Notably, only HTRA1 was able to penetrate the cell membrane of MG-63 cells in dissolved form. However, the application of endocytosis inhibitors revealed that the uptake pathway was different for dissolved HTRA1 and HTRA1-loaded nanoparticles.
RESUMO
The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.
Assuntos
DNA/química , Proteínas de Choque Térmico/química , Ligantes , Proteínas Periplásmicas/química , Domínios e Motivos de Interação entre Proteínas , Serina Endopeptidases/química , Sítios de Ligação , Técnicas de Química Sintética , Engenharia Genética , Proteínas de Choque Térmico/genética , Modelos Moleculares , Imagem Molecular , Sondas Moleculares , Estrutura Molecular , Proteínas Periplásmicas/genética , Polímeros/química , Serina Endopeptidases/genéticaRESUMO
The protease-chaperone DegP undergoes secondary through quaternary structural changes, regulating function and preventing indiscriminate proteolysis. Several structures of DegP oligomers have been observed, including the resting state 6-mer and the 12-mer and 24-mer active states. However, the precise events of the transition between the resting and active states still need to be elucidated. We used native mass spectrometry to demonstrate that binding of multiple substrate-mimicking peptide ligands to the DegP resting state occurs prior to the transition to an active conformation. This transition occurred at a 6-mer occupancy of 40% for each peptide ligand. We observed ligand-specific 9-mer formation with a maximum load of 9 peptides, whereas other substrates led to 12-mers accommodating 24 peptides. Based on these data, we present a model for the initial steps of substrate-induced transitions from the resting to active states of DegP.
Assuntos
Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Proteínas Periplásmicas/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Especificidade por SubstratoRESUMO
Allostery is a fundamental regulatory mechanism that is based on a functional modulation of a site by a distant site. Allosteric regulation can be triggered by binding of diverse allosteric effectors, ranging from small molecules to macromolecules, and is therefore offering promising opportunities for functional modulation in a wide range of applications including the development of chemical probes or drug discovery. Here, we provide an overview of key classes of allosteric protease effectors, their corresponding molecular mechanisms, and their practical implications.
Assuntos
Peptídeo Hidrolases , Regulação Alostérica/efeitos dos fármacos , Descoberta de Drogas , Humanos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/farmacologiaRESUMO
Channeling of misfolded proteins into repair, assembly or degradation pathways is often mediated by complex and multifunctional cellular factors. Despite detailed structural information, the underlying regulatory mechanisms governing these factors are not well understood. The extracytoplasmic heat-shock factor DegP (HtrA) is a well-suited model for addressing mechanistic issues, as it is regulated by the common mechanisms of allostery and activation by oligomerization. Site-directed mutagenesis combined with refolding and oligomerization studies of chemically denatured DegP revealed how substrates trigger the conversion of the resting conformation into the active conformation. Binding of specific peptides to PDZ domain-1 causes a local rearrangement that is allosterically transmitted to the substrate-binding pocket of the protease domain. This activated state readily assembles into larger oligomeric particles, thus stabilizing the catalytically active form and providing a degradation cavity for protein substrates. The implications of these data for the mechanism of protein quality control are discussed.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas Periplásmicas/química , Proteínas Periplásmicas/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Bactérias/genética , Proteínas de Bactérias/genética , Domínio Catalítico , Proteínas de Choque Térmico/genética , Modelos Moleculares , Domínios PDZ , Peptídeos/química , Peptídeos/metabolismo , Proteínas Periplásmicas/genética , Mutação Puntual , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Serina Endopeptidases/genéticaRESUMO
Two members of the widely conserved HtrA family of serine proteases, DegP and DegS, are key players in extracytoplasmic protein quality control. The underlying mechanisms of their main functions in stress sensing, regulation and protection during the unfolded protein response are discussed.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas Periplásmicas/química , Proteínas Periplásmicas/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão GênicaRESUMO
Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.
Assuntos
Proteínas de Bactérias/metabolismo , Haemophilus influenzae/enzimologia , NAD/biossíntese , Niacinamida/análogos & derivados , Pentosiltransferases/fisiologia , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Membrana Celular/metabolismo , Haemophilus influenzae/genética , Mutação , Niacinamida/metabolismo , Nicotinamida Fosforribosiltransferase , Fosforilação , Fosfotransferases/genética , Transporte Proteico , Compostos de Piridínio , RibonucleotídeosRESUMO
The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized for Haemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC. pnuC of H. influenzae is only distantly related to pnuC of Escherichia coli and Salmonella enterica serovar Typhimurium. When E. coli PnuC was expressed in an H. influenzae pnuC mutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory for H. influenzae growth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory to H. influenzae and is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in a pnuC+ background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistant H. influenzae isolates were characterized, and mutations in nadR could be detected. We also tested other species of the family Pasteurellaceae, Pasteurella multocida and Actinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.