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1.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1689-1698, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924367

RESUMO

Enterokinase is a class of serine proteases that specifically recognize the cleavage DDDDK sequences. Therefore, enterokinase has been widely used as a tool enzyme in the field of biomedicine. Currently, the expression level of enterokinase in Pichia pastoris is low, which hinders related practical applications. In this study, the effects of six different signal peptides SP1, SP2, SP3, SP4, SP7 and SP8 on the secretory expression of enterokinase in Pichia pastoris were studied. Compared with α-factor, SP1 significantly increased the secretory expression of enterokinase (from 6.8 mg/L to 14.3 mg/L), and the enterokinase activity increased from (2 390±212) U/mL to (4 995±378) U/mL in shaking flask cultures. On this basis, the enterokinase activity was further enhanced to (7 219±489) U/mL by co-expressing the endogenous protein Kex2. Moreover, the activity that the mutant strain with N-terminal fusion of three amino acids of WLR was increased to (15 145±920) U/mL with a high specific activity of (1 174 600±53 100) U/mg. The efficient secretory expression of enterokinase laid a foundation for its applications in near future.


Assuntos
Enteropeptidase , Regulação Fúngica da Expressão Gênica , Microbiologia Industrial , Pichia , Aminoácidos , Enteropeptidase/genética , Regulação Fúngica da Expressão Gênica/genética , Microbiologia Industrial/métodos , Pichia/enzimologia , Pichia/genética , Sinais Direcionadores de Proteínas
2.
Proc Natl Acad Sci U S A ; 117(29): 16938-16948, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616570

RESUMO

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.


Assuntos
Toxina Diftérica/metabolismo , Endopeptidases/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteólise , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Antineoplásicos/uso terapêutico , Células Cultivadas , Toxina Diftérica/química , Toxina Diftérica/genética , Endopeptidases/química , Endopeptidases/genética , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/uso terapêutico , Proteínas ras/genética
3.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641482

RESUMO

C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry.IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.


Assuntos
Antígenos de Superfície/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus/fisiologia , Interações Hospedeiro-Patógeno , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Internalização do Vírus , Sequência de Aminoácidos , Anfotericina B/farmacologia , Betacoronavirus/efeitos dos fármacos , Linhagem Celular , Coronavirus/efeitos dos fármacos , Infecções por Coronavirus/epidemiologia , Suscetibilidade a Doenças , Evolução Molecular , Proteínas Ligadas por GPI/metabolismo , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Sinais Direcionadores de Proteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(32): 19399-19407, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719124

RESUMO

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/genética
5.
Nat Commun ; 11(1): 3167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576831

RESUMO

Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific CEP gene, MtCEP7, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor. MtCEP7 and MtCLE13 expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates MtCEP7 and MtCLE13, and a NIN Binding Site (NBS) identified within the proximal MtCEP7 promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.


Assuntos
Peptídeos/química , Nodulação/fisiologia , Rhizobium/metabolismo , Fatores de Transcrição/metabolismo , Citocininas/metabolismo , Epiderme , Regulação da Expressão Gênica de Plantas , Lotus/metabolismo , Medicago truncatula , Peptídeos/genética , Proteínas de Plantas , Nodulação/genética , Raízes de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas Quinases , Sinais Direcionadores de Proteínas/genética , Nódulos Radiculares de Plantas , Sinorhizobium meliloti/metabolismo , Simbiose
6.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1223-1231, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597072

RESUMO

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.


Assuntos
Anticorpos Antivirais , Citomegalovirus , Imunoglobulina M , Proteínas Recombinantes de Fusão , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Cricetinae , Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Imunoglobulina M/imunologia , Camundongos , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/imunologia
7.
Nucleic Acids Res ; 48(12): 6491-6502, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484544

RESUMO

Multifunctional proteins often perform their different functions when localized in different subcellular compartments. However, the mechanisms leading to their localization are largely unknown. Recently, 3'UTRs were found to regulate the cellular localization of newly synthesized proteins through the formation of 3'UTR-protein complexes. Here, we investigate the formation of 3'UTR-protein complexes involving multifunctional proteins by exploiting large-scale protein-protein and protein-RNA interaction networks. Focusing on 238 human 'extreme multifunctional' (EMF) proteins, we predicted 1411 3'UTR-protein complexes involving 54% of those proteins and evaluated their role in regulating protein cellular localization and multifunctionality. We find that EMF proteins lacking localization addressing signals, yet present at both the nucleus and cell surface, often form 3'UTR-protein complexes, and that the formation of these complexes could provide EMF proteins with the diversity of interaction partners necessary to their multifunctionality. Our findings are reinforced by archetypal moonlighting proteins predicted to form 3'UTR-protein complexes. Finally, the formation of 3'UTR-protein complexes that involves up to 17% of the proteins in the human protein-protein interaction network, may be a common and yet underestimated protein trafficking mechanism, particularly suited to regulate the localization of multifunctional proteins.


Assuntos
Regiões 3' não Traduzidas , Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Humanos , Proteínas de Membrana/química , Ligação Proteica , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas , Transporte Proteico , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química
8.
Nat Commun ; 11(1): 2355, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398688

RESUMO

Correct intracellular distribution of proteins is critical for the function of eukaryotic cells. Certain proteins are targeted to more than one cellular compartment, e.g. to mitochondria and peroxisomes. The protein phosphatase Ptc5 from Saccharomyces cerevisiae contains an N-terminal mitochondrial presequence followed by a transmembrane domain, and has been detected in the mitochondrial intermembrane space. Here we show mitochondrial transit of Ptc5 to peroxisomes. Translocation of Ptc5 to peroxisomes depended both on the C-terminal peroxisomal targeting signal (PTS1) and N-terminal cleavage by the mitochondrial inner membrane peptidase complex. Indirect targeting of Ptc5 to peroxisomes prevented deleterious effects of its phosphatase activity in the cytosol. Sorting of Ptc5 involves simultaneous interaction with import machineries of both organelles. We identify additional mitochondrial proteins with PTS1, which localize in both organelles and can increase their physical association. Thus, a tug-of-war-like mechanism can influence the interaction and communication of two cellular compartments.


Assuntos
Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Sinais Direcionadores de Proteínas , Saccharomyces cerevisiae/citologia
9.
Nucleic Acids Res ; 48(12): 6726-6739, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449932

RESUMO

Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen-receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG-12RSS-23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG-RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.


Assuntos
Conformação de Ácido Nucleico , Sinais Direcionadores de Proteínas/genética , Receptores de Antígenos/genética , Recombinação V(D)J/genética , Animais , Clivagem do DNA , Linfócitos/metabolismo , Imagem Individual de Molécula , Vertebrados/genética , Vertebrados/crescimento & desenvolvimento
10.
Arch Microbiol ; 202(7): 1669-1675, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32285165

RESUMO

Archaea swim using archaella that are domain-specific rotary type IV pilus-like appendages. The structural components of the archaellum filament are archaellins, initially made as preproteins with type IV pilin-like signal peptides which are removed by signal peptidases that are homologues of prepilin peptidases that remove signal peptides from type IV pilins. N-terminal sequences of archaellins, including the signal peptide cleavage site, are conserved and various positions have been previously shown to be critical for signal peptide removal. Archaellins have an absolute conservation of glycine at the + 3 position from the signal peptide cleavage site. To investigate its role in signal peptide cleavage, I used archaellin variants in which the + 3 glycine was mutated to all other possibilities in in vitro cleavage reactions. Cleavage was observed with ten different amino acids at the + 3 position, indicating that the observed glycine conservation is not required for this essential processing step.


Assuntos
Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Mathanococcus/enzimologia , Mathanococcus/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas Arqueais/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/enzimologia , Mathanococcus/metabolismo , Sinais Direcionadores de Proteínas
11.
Int J Med Microbiol ; 310(2): 151396, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32005588

RESUMO

The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected ß-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to ß-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem.


Assuntos
Cefoperazona/farmacologia , Cefalosporinas/farmacologia , Periplasma/enzimologia , Serina Endopeptidases/genética , Sphingomonadaceae/efeitos dos fármacos , Fatores de Transcrição/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Periplasma/efeitos dos fármacos , Sinais Direcionadores de Proteínas , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética
12.
J Basic Microbiol ; 60(4): 341-350, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32043631

RESUMO

Vacuoles are useful materials with antimicrobial and anticancerous properties. Vacuolar proteins can discompose macromolecules from the outside of yeast cells. The objective of this study was to determine the function of a protein transported into a vacuole. Specifically, cytosolic protein aldehyde dehydrogenase 6 (ALD6) was used for the delivery to the vacuole. To transport cytosolic protein to the vacuole in this study, a transfer vector including a signal peptide sequence isolated from vacuolar protein proteinase A was designed. A signal peptide is an amino acid sequence in front of the transported protein. Signal peptides have various delivery pathways according to the kind of signal sequence they contain. They play important roles in transporting proteins to organelles, in cellular mechanisms, and the transfer of protein outside and inside eukaryotes. Thus, we focused on the design of a transfer vector containing a signal peptide sequence isolated from the DNA sequence of proteinase A (PEP4). In addition, this study evaluated the expression level of cytosolic ALD6 after being transported into the yeast vacuole. Our results showed that the developed transfer vector was useful for delivering proteins to vacuole by using signal peptide sequence. Therefore, this transfer vector might be used as a tool to deliver target proteins to organelles of interest in eukaryotes.


Assuntos
Aldeído Oxirredutases/metabolismo , Ácido Aspártico Endopeptidases/genética , Citosol/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Vacúolos/metabolismo , Aldeído Oxirredutases/genética , Transporte Proteico
13.
Biochem Biophys Res Commun ; 524(3): 555-560, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32014252

RESUMO

The New Delhi metallo-ß-lactamase (NDM-1) mediates resistance to ß-lactam antibiotics. NDM-1 was likely formed as the result of a gene fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-ß -lactamase. We show that NDM-1 has an atypical signal peptide and is inefficiently secreted. Two new blaNDM-1 alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A) were studied. Here, we show that both the P9R and P28A substitutions improve secretion compared to NDM-1 and display higher resistance to some ß-lactam antibiotics. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. For NDM-1, we detected a signal peptide cleavage site between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. In addition, expression of NDM-2 improves the fitness of E. coli, compared to NDM-1, in the absence of antibiotic selection. This study shows how optimization of the secretion efficiency of NDM-1 leads to increased resistance and increased fitness.


Assuntos
Alelos , Evolução Molecular , Aptidão Genética , Klebsiella/enzimologia , Klebsiella/genética , Seleção Genética , beta-Lactamases/genética , Sequência de Aminoácidos , Animais , Resistência Microbiana a Medicamentos/genética , Camundongos , Testes de Sensibilidade Microbiana , Sinais Direcionadores de Proteínas , beta-Lactamases/química
14.
Gene ; 737: 144435, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32044407

RESUMO

The cancer-brake gene CTLA4 has a vital function in suppressing the immune responses of activated T lymphocytes. Numerous reports explored the impact of various CTLA4 variants with the predisposition for malignancies but with unconvincing findings. Hence, this study is designed to assess the association of CTLA4 (c.49A>G, rs231775) variant with the outcome of breast carcinoma. A total of 272 participants (93 BC patients and 179 cancer-free healthy volunteers) were enrolled. Genomic DNA for all participants was genotyped for CTLA4 (c.49A>G) variant via TaqMan genotyping assay. Patients with A/G genotype conferred protection against developing BC under heterozygote comparison (OR = 0.56, 95%CI = 0.31-0.98) as well dominant model (OR = 0.55, 95%CI = 0.32-0.97). AG/GG genotypes were anchored with an increased risk of nodal infiltration (OR = 2.90, 95%CI = 1.03-8.17, P = 0.037), metastasis (OR = 4.46, 95%CI = 1.18-16.8, P = 0.019), advanced clinical stage (OR = 6.54, 95%CI = 2.06-20.75, P < 0.001), recurrence (OR = 5.2, 95%CI = 1.73-15.7, P = 0.001), and shorter survival (OR = 2.54, 95%CI = 1.08-5.99, P = 0.032). In addition, functional enrichment analysis revealed the key role of CTLA4 in cancer immunosurveillance. Our findings indicated that the CTLA4 c.49A>G variant might have prognostic as well diagnostic impact in breast cancer.


Assuntos
Neoplasias da Mama/genética , Antígeno CTLA-4/genética , Mutação de Sentido Incorreto , Sinais Direcionadores de Proteínas , Adulto , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Egito , Feminino , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico
15.
PLoS One ; 15(2): e0222685, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32078628

RESUMO

Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that there is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.


Assuntos
Sinais Direcionadores de Proteínas/fisiologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Animais , Células COS , Chlorocebus aethiops , Endopeptidases/metabolismo , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Inibidores de Proteases/farmacologia , Receptor PAR-2/genética , Receptores de Superfície Celular , Tripsina/metabolismo
16.
Appl Microbiol Biotechnol ; 104(4): 1621-1632, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31907577

RESUMO

Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (PAOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters PGAP, PGAP(m), and PTEF1 and signal peptides α-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 × 105 U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 × 105 U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 × 105 U/mL. Finally, LHyal activity of 2.12 × 106 U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 × 104 U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.


Assuntos
Hialuronoglucosaminidase/biossíntese , Sanguessugas/enzimologia , Pichia/metabolismo , Animais , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Hialuronoglucosaminidase/genética , Microbiologia Industrial , Sanguessugas/genética , Pichia/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Fatores de Transcrição/genética
17.
Malar J ; 19(1): 29, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952536

RESUMO

BACKGROUND: Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding proteins (OBPs), which influence the chemosensory system and behavioural responses, is essential to understanding the population structure and developing effective control measures against this vector. The present study was designed to identify and analyse the obp1 gene in An. maculipennis. METHODS: Adults of An. maculipennis sensu stricto were collected in Zanjan Province, northwest of Iran, and gDNAs of female mosquitoes were extracted. Fragments of An. maculipennis obp1 (Amacobp1) gene were amplified using degenerate and specific primers, and some of amplicons were selected for sequencing. RESULTS: Analysis of amplified products identified that the sequence of Amacobp1 gene was 1341 bp long. This gene contains three exons (5', internal, and 3'of 160, 256, and 18 bp, respectively) and encodes 144 amino acids. The sizes of introns I and II in deduced gene are 268 and 358 nucleotides, respectively. The amino acid sequence in the C-terminal of AmacOBP1 is similar to that of major malaria vector Anopheles species. However, its N-terminal has a specific signal peptide with 19 amino acids. This peptide is conserved in different studied populations, and its sequence of amino acids shows the most variation among anopheline species. CONCLUSIONS: Degenerate primers in this study are suggested for studying obp1 gene in Anopheles species. Amacobp1 gene is proposed as a molecular marker for the detection of intraspecific ecotypes and diagnosis of different species within Maculipennis Group. Moreover, the N-terminal of AmacOBP1 peptide is recommended as a molecular marker to identify the Amacobp1 expression patterns in different chemosensory organs for assessing the molecular mechanisms and developing novel behavioural disturbance agents to control An. maculipennis.


Assuntos
Anopheles/química , Mosquitos Vetores/química , Receptores Odorantes/genética , Sequência de Aminoácidos , Animais , Anopheles/classificação , Anopheles/genética , Sequência de Bases , DNA/química , DNA/genética , DNA/isolamento & purificação , Éxons , Feminino , Íntrons , Irã (Geográfico) , Masculino , Mosquitos Vetores/classificação , Mosquitos Vetores/genética , Filogenia , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Receptores Odorantes/química , Alinhamento de Sequência
18.
Appl Microbiol Biotechnol ; 104(3): 1201-1209, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900564

RESUMO

We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 1011 pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10 ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5 days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.


Assuntos
Bacteriófago M13/genética , Proteínas do Capsídeo/genética , Técnicas de Visualização da Superfície Celular/métodos , Escherichia coli/genética , Ácidos Nucleicos Imobilizados , Clonagem Molecular , Mutação , Oligopeptídeos , Biblioteca de Peptídeos , Plasmídeos/genética , Sinais Direcionadores de Proteínas/genética
19.
Microb Cell Fact ; 19(1): 11, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964372

RESUMO

BACKGROUND: In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. RESULTS: High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. CONCLUSIONS: We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.


Assuntos
Proteínas de Bactérias/biossíntese , Técnicas Biossensoriais , Corynebacterium glutamicum/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Engenharia Genética , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo , Via Secretória , Proteínas de Transporte Vesicular/metabolismo
20.
Prep Biochem Biotechnol ; 50(2): 141-147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31647371

RESUMO

Activin A is a member of the transforming growth factor-beta (TGF-ß) protein superfamily, which acts as a hormone in regulating cell proliferation and differentiation. Structurally, activin is a dimer of two subunits linked by a disulfide bond. Since the correct folding of this protein is essential for its function, we aimed to use a modified signal peptide to target the expressed recombinant protein to the periplasm of Escherichia coli as an effective strategy to produce correctly-folded activin A. Therefore, the coding sequence of native Iranian Bacillus licheniformis α-amylase signal peptide was modified and its efficiency was checked by SignalP bioinformatics tool. Then its final sequence was cloned upstream of the activin A mature cDNA. Protein expression was done using 1 mM of isopropyl thio-ß-D-galactoside (IPTG) and a post-induction time of 8 hr. Additionally, following purification of recombinant activin A, circular dichroism spectroscopy was used to determine the accuracy of secondary structure of the protein. Importantly, differentiation of K562 cells to the red blood cell was confirmed by measuring the amount of Fe+2 ions after treatment with recombinant activin A. The results indicated that the produced recombinant activin A had the same secondary structure as the commercial human activin A and was fully functional.


Assuntos
Ativinas/genética , Escherichia coli/genética , Periplasma/metabolismo , Sinais Direcionadores de Proteínas , Ativinas/química , Ativinas/isolamento & purificação , Cromatografia de Afinidade , Humanos , Células K562 , Estrutura Secundária de Proteína , alfa-Amilases/metabolismo
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