RESUMO
Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)-dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h-region are critical for sensitivity to CADA. In particular, exchanging Gln-15, Val-17 or Pro-20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N-terminal portion of the mature protein, these residues mediate full susceptibility to the co-translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h-domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co-translational translocation inhibitor.
Assuntos
Antígenos CD4 , Sinais Direcionadores de Proteínas , Inibidores da Síntese de Proteínas , Antígenos CD4/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sinais Direcionadores de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
Proteins routed to the secretory pathway start their journey by being transported across biological membranes, such as the endoplasmic reticulum. The essential nature of this protein translocation process has led to the evolution of several factors that specifically target the translocon and block translocation. In this review, various translocation pathways are discussed together with known inhibitors of translocation. Properties of signal peptide-specific systems are highlighted for the development of new therapeutic and antimicrobial applications, as compounds can target signal peptides from either host cells or pathogens and thereby selectively prevent translocation of those specific proteins. Broad inhibition of translocation is also an interesting target for the development of new anticancer drugs because cancer cells heavily depend on efficient protein translocation into the endoplasmic reticulum to support their fast growth.
Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Retículo Endoplasmático/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Animais , Retículo Endoplasmático/metabolismo , Humanos , Modelos Moleculares , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Sinais Direcionadores de Proteínas/efeitos dos fármacosRESUMO
The small molecule CADA was shown to down-modulate the expression of human CD4 in a signal peptide-dependent way through inhibition of its cotranslational translocation across the ER membrane. Previous studies characterizing general glycoprotein levels and the expression of 14 different cell surface receptors showed selectivity of CADA for human CD4. Here, a PowerBlot Western Array was used as a screen to analyze the proteome of CADA-treated SUP-T1 human CD4+ T lymphocytes. This high-throughput monoclonal antibody panel-based immunoblotting assay of cellular signaling proteins revealed that only a small subset of the 444 detected proteins was differentially expressed after treatment with CADA. Validation of these proteomic data with optimized immunoblot analysis confirmed the CADA-induced change in expression of the cell cycle progression regulator pRb2 and the transcription factor c-Jun. However, the up-regulation of pRb2 or down-modulation of c-Jun by CADA had no impact on cell cycle transition. Also, the reduced protein level of human CD4 did not inhibit T cell receptor signaling. Interestingly, the signal peptide-containing membrane protein sortilin was identified as a new substrate for CADA. Both cellular expression and in vitro cotranslational translocation of sortilin were significantly reduced by CADA, although to a lesser extent as compared with human CD4. Our data demonstrate that a small signal peptide-binding drug is able to down-modulate the expression of human CD4 and sortilin, apparently with low impact on the cellular proteome.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Proteômica/métodos , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Transporte Proteico/efeitos dos fármacosRESUMO
Cyclotriazadisulfonamide prevents HIV entry into cells by down-modulating surface CD4 receptor expression through binding to the CD4 signal peptide. According to a two-site binding model, 28 new unsymmetrical analogues bearing a benzyl tail group and nine bearing a cyclohexylmethyl tail have been designed and synthesized. The most potent new CD4 down-modulator (40 (CK147); IC50 63 nM) has a 4-dimethylaminobenzenesulfonyl side arm. One of the two side arms was varied with substituents in different positions. This gave a range of CD4 down-modulation potencies that correlated well with anti-HIV-1 activities. The side arms of 21 of the new benzyl-tailed analogues were modeled by means of quantum mechanical calculations. For CADA analogues with arenesulfonamide side arms, the pIC50 values for CD4 down-modulation correlated with the component of the electric dipole moment in the aromatic ring, suggesting that an attractive electronic interaction is a major factor determining the stability of the complex between the molecule and its target.
Assuntos
Antígenos CD4/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacologia , Compostos Heterocíclicos com 1 Anel/síntese química , Compostos Heterocíclicos com 1 Anel/farmacologia , Transporte Proteico/efeitos dos fármacos , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Animais , Células CHO , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Regulação para Baixo/efeitos dos fármacos , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Sinais Direcionadores de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Sequência de Aminoácidos , Antígenos CD4/química , Antígenos CD4/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Conformação Proteica , Transporte Proteico/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Importance: Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins--enzymes derived from bacterial viruses--represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.