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1.
J Biosci Bioeng ; 136(3): 173-181, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37487915

RESUMO

Cancer treatment has been revolutionized by immune checkpoint inhibitors, which regulate immune cell function by blocking the interactions between immune checkpoint molecules and their ligands. The interaction between programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a target for immune checkpoint inhibitors. Nanobodies, which are recombinant variable domains of heavy-chain-only antibodies, can replace existing immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 conventional antibodies. However, the screening process for high-affinity nanobodies is laborious and time-consuming. Here, we identified high-affinity anti-PD-1 nanobodies using peptide barcoding, which enabled reliable and efficient screening by distinguishing each nanobody with a peptide barcode that was genetically appended to each nanobody. We prepared a peptide-barcoded nanobody (PBNb) library with thousands of variants. Three high-affinity PBNbs were identified from the PBNb library by quantifying the peptide barcodes derived from high-affinity PBNbs. Furthermore, these three PBNbs neutralized the interaction between PD-1 and PD-L1. Our results demonstrate the utility of peptide barcoding and the resulting nanobodies can be used as experimental tools and antitumor agents.


Assuntos
Antineoplásicos , Anticorpos de Domínio Único , Anticorpos de Domínio Único/química , Inibidores de Checkpoint Imunológico , Peptídeos/química , Biblioteca de Peptídeos
2.
iScience ; 25(2): 103675, 2022 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-35141499

RESUMO

Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.

3.
Sci Rep ; 11(1): 21516, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728738

RESUMO

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence-function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody-antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence-function relationships.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Fragmentos de Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Domínio Único/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Ligação Proteica , Proteômica , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia
4.
Sci Rep ; 11(1): 11059, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040114

RESUMO

Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.


Assuntos
Membrana Celular , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/genética , Proteínas/química
5.
Cell Signal ; 68: 109542, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31954176

RESUMO

The target of rapamycin complex 1 (TORC1) protein kinase is activated by nutrients and controls nutrient uptake via the membrane trafficking of various nutrient permeases. However, its molecular mechanisms remain elusive. Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, "lipid rafts", which are critical for intracellular protein sorting. Here we show that a novel target of rapamycin (TOR)-interacting transcriptional activator Vhr2 is required for full expression of some ERG genes for ergosterol biogenesis and for proper sorting of the tryptophan permease Tat2 in budding yeast. Loss of Vhr2 caused sterol biogenesis disturbance and Tat2 missorting. TORC1 activity maintained VHR2 transcript and protein levels, and total sterol levels. Vhr2 was not involved in regulation of the TORC1-downstream protein kinase Npr1, which regulates Tat2 sorting. This study suggests that TORC1 regulates nutrient uptake via sterol biogenesis.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimologia , Transativadores/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Triptofano/metabolismo , Regulação Fúngica da Expressão Gênica , Ligação Proteica , Transporte Proteico , Proteólise , Saccharomycetales/genética , Esteróis/biossíntese , Ubiquitinação , Regulação para Cima/genética , Vacúolos/metabolismo
6.
J Vet Med Sci ; 80(11): 1782-1786, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30282841

RESUMO

The purpose of this study was to detect porcine epidemic diarrhea virus (PEDV) subclinically infected pigs shipped from non-case farms to slaughterhouses. Systematic sampling was conducted at two slaughterhouses. A total of 1,556 blood samples were collected from 80 case and non-case farms from pigs over 6 months old. Blood samples were centrifuged to obtain sera. Serial serum dilutions were subjected to serological examination for PEDV presence using Neutralization test (NT). The cut-off titer was set at titer of 1:2 dilution and farms with at least one positive sample in duplicate were classified as PED-positive farms. Several non-case farms (9.4%, 6/64) and 100% (16/16) of the case farms were indeed positive for PEDV. The proportion of seropositive animals from case farms was 63.7%, significantly different from that of non-case farms (4.3%, P<0.05). In both case and non-case farms, the proportion of seropositive animals in farrow-to-finish farms was significantly higher than in wean-to-finish farms (P<0.05). Seropositive animals in non-case farms were detected by NT in a sero-survey by sampling at slaughterhouses. Therefore, subclinically infected pigs should be considered prior to shipment.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/imunologia , Doenças dos Suínos/virologia , Animais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Japão/epidemiologia , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/imunologia
7.
J Cell Biol ; 217(8): 2675-2690, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29959231

RESUMO

Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear-vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA-nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.


Assuntos
DNA Ribossômico/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia/fisiologia , Sobrevivência Celular , Imunoprecipitação da Cromatina , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
8.
EuroIntervention ; 14(8): 898-906, 2018 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-29688181

RESUMO

AIMS: Fractional flow reserve (FFR), assessed using distal coronary pressure/aortic pressure (Pd)/(Pa) ratio, functionally evaluates coronary stenosis. An assessment method without vasodilators would be helpful. A single intracoronary bolus of saline decreases Pd because of the speculated low-viscosity effect. We hypothesised that saline-induced Pd/Pa ratio (SPR) could functionally evaluate coronary stenosis. This study aimed to test the accuracy and utility of SPR for predicting FFR ≤0.80. METHODS AND RESULTS: In 137 coronary lesions with over 50% angiographic diameter stenosis, SPR was assessed using an intracoronary bolus of saline (2 mL/s) for five heartbeats (SPR-5) and three heartbeats (SPR-3). FFR was obtained after intravenous adenosine infusion (140 µg/kg/min). There was a strong correlation between FFR and SPR-5 or SPR-3 (R=0.941 and R=0.933, respectively). Receiver operating characteristic (ROC) curve analysis demonstrated good accuracy (86.3%) for SPR-5, with a cut-off of ≤0.84 for predicting FFR ≤0.80 (area under ROC curve 0.96, specificity 94.3, sensitivity 79.9). Thirty-three lesions (24%) were located in the "grey zone" (SPR 0.83-0.88). No complications were observed in 673 SPR measurements. CONCLUSIONS: SPR may accurately predict FFR and can limit adenosine use to one in four lesions. Further studies are needed to confirm the validity of SPR.


Assuntos
Estenose Coronária , Reserva Fracionada de Fluxo Miocárdico , Cateterismo Cardíaco , Angiografia Coronária , Vasos Coronários , Humanos , Paládio , Valor Preditivo dos Testes , Protoactínio , Curva ROC , Índice de Gravidade de Doença
9.
Curr Genet ; 64(4): 907-917, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29423676

RESUMO

For maintenance of cytoplasmic protein quality control (PQC), cytoplasmic heat shock proteins (HSPs) negatively control heat shock transcriptional factor (HSF) in a negative feedback loop. However, how mitochondrial protein quality control (mtPQC) is maintained is largely unknown. Here we present evidence that HSF directly monitors mtPQC in the budding yeast Saccharomyces cerevisiae. Mitochondrial HSP70 (Ssc1) negatively regulated HSF activity. Importantly, HSF was localized not only in the nucleus but also on mitochondria. The mitochondrial localization of HSF was increased by heat shock and compromised by SSC1 overexpression. Furthermore, the mitochondrial protein translocation system downregulated HSF activity. Finally, mtPQC modulated the mtHSP genes SSC1 and MDJ1 via HSF, and SSC1 overexpression compromised mitochondrial function. These findings illustrate a model in which HSF directly monitors mtPQC.


Assuntos
ATPases Transportadoras de Cálcio/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Membrana/genética , Mitocôndrias/genética , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sítios de Ligação , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição de Choque Térmico/genética , Ligação Proteica , Resposta a Proteínas não Dobradas/genética
10.
Biosci Biotechnol Biochem ; 81(2): 307-310, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27659307

RESUMO

Autophagic degradation of ribosomes is promoted by nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1). Here we show that selective autophagic degradation of ribosomes (called ribophagy) after TORC1 inactivation requires the specific autophagy receptor Atg11. Rim15 protein kinase upregulated ribophagy, while it downregulated non-selective degradation of ribosomes.


Assuntos
Autofagia , Proteínas Quinases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas Relacionadas à Autofagia/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo
11.
PLoS One ; 11(12): e0166636, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27973551

RESUMO

Target of rapamycin complex 1 (TORC1) phosphorylates autophagy-related Atg13 and represses autophagy under nutrient-rich conditions. However, when TORC1 becomes inactive upon nutrient depletion or treatment with the TORC1 inhibitor rapamycin, Atg13 dephosphorylation occurs rapidly, and autophagy is induced. At present, the phosphatases involved in Atg13 dephosphorylation remain unknown. Here, we show that two protein phosphatase 2A (PP2A) phosphatases, PP2A-Cdc55 and PP2A-Rts1, which are activated by inactivation of TORC1, are required for sufficient Atg13 dephosphorylation and autophagy induction after TORC1 inactivation in budding yeast. After rapamycin treatment, dephosphorylation of Atg13, activation of Atg1 kinase, pre-autophagosomal structure (PAS) formation and autophagy induction are all impaired in PP2A-deleted cells. Conversely, overexpression of non-phosphorylatable Atg13 suppressed defects in autophagy in PP2A mutant. This study revealed that the orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Autofagossomos , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Sirolimo/química
12.
Biosci Biotechnol Biochem ; 80(3): 473-8, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26523765

RESUMO

The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase ESP1 in the budding yeast Saccharomyces cerevisiae. All mutants were defective in sister chromatid separation at the restricted temperature. Some esp1-ts mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some esp1-ts mutants, whereas it rather exacerbated it in others. The Drosophila Pumilio homolog MPT5 was isolated as a high-dosage suppressor of esp1-ts cells. We discuss various features of separase based on these findings.


Assuntos
Mutação , Saccharomyces cerevisiae/enzimologia , Separase/genética , Benomilo/farmacologia , Bleomicina/farmacologia , Proteínas de Fluorescência Verde/genética , Pressão Osmótica , Saccharomyces cerevisiae/efeitos dos fármacos , Separase/metabolismo , Temperatura
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