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1.
Sci Rep ; 12(1): 12297, 2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35853959

RESUMO

Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes. We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury. A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods. Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data, Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8, -9 and -3, suggesting that rituximab may inhibit apoptosis. In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes.


Assuntos
Síndrome Nefrótica , Podócitos , Apoptose , Células Cultivadas , Humanos , Imunossupressores/metabolismo , Imunossupressores/farmacologia , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Puromicina/farmacologia , Puromicina Aminonucleosídeo/metabolismo , Puromicina Aminonucleosídeo/farmacologia , Rituximab/metabolismo , Rituximab/farmacologia
2.
STAR Protoc ; 3(2): 101436, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35693210

RESUMO

Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Embrionárias Murinas , Animais , Sistemas CRISPR-Cas/genética , Células-Tronco Embrionárias , Camundongos , Puromicina/farmacologia , Transfecção
3.
Asian Pac J Cancer Prev ; 23(5): 1803-1812, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35633567

RESUMO

Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin. OBJECTIVE: This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing. METHODS: Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment. RESULTS: The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL. CONCLUSION: Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.


Assuntos
Antineoplásicos , Neoplasias de Mama Triplo Negativas , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Cetuximab/farmacologia , Receptores ErbB/metabolismo , Humanos , Puromicina/farmacologia , Puromicina/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo
4.
Stem Cell Res Ther ; 13(1): 131, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-35346349

RESUMO

BACKGROUND: Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0V) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0V INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0V INs. This study generated a transgenic mESC line to enrich V0V INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies. METHODS: The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0V IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0V INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs). RESULTS: V0V IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting. CONCLUSIONS: Evx1-PAC mESCs can be used to purify V0V IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0V INs.


Assuntos
Interneurônios , Células-Tronco Embrionárias Murinas , Animais , Proteínas de Homeodomínio/genética , Humanos , Interneurônios/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Puromicina/metabolismo , Puromicina/farmacologia
5.
PLoS One ; 17(3): e0265183, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35333876

RESUMO

Transgenic proteins can be routinely expressed in various mammalian cell types via different transgenic systems, but the efficiency of transgene expression is constrained by the complex interplay among factors such as the temporal consistency of expression and compatibility with specific cell types, including ocular cells. Here, we report a more efficient way to express an enhanced green fluorescent protein (EGFP) in human corneal fibroblasts, corneal epithelial cells, and conjunctival epithelial cells through a lentiviral expression system. The relative transducing unit criterion for EGFP-expressing pseudovirions was first determined in HEK-293T cells. Homogeneous populations of EGFP-positive and EGFP-negative cells could be isolated by cell sorting. The half-maximal inhibitory concentration (IC50) value for puromycin was calculated according to viability curves for each cell type. The results revealed that cell types differed with respect to EGFP expression efficiency after transduction with the same amount of EGFP-encoding pseudovirions. Using a cell sorter, the homogeneity of EGFP-positive cells reached >95%. In the initial sorting stage, however, the efficiency of EGFP expression in the sorted cells was noticeably reduced after two rounds of sequential culture, but repeated sorting for up to four rounds yielded homogeneous EGFP-positive human corneal fibroblasts that could be maintained in continuous culture in vitro. The sorted EGFP-positive cells retained their proper morphology and cell type-specific protein expression patterns. Puromycin resistance was found to depend on cell type, indicating that the IC50 for puromycin must be determined for each cell type to ensure the isolation of homogeneous EGFP-positive cells. Taken together, repeated cell sorting is an efficient means of obtaining homogeneous populations of ocular cells expressing a transgenic protein during continuous culture without the potential confounding effects of antibiotics.


Assuntos
Mamíferos , Animais , Animais Geneticamente Modificados , Separação Celular , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mamíferos/metabolismo , Puromicina/farmacologia , Transgenes
6.
Methods Mol Biol ; 2428: 63-73, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35171473

RESUMO

The attenuation of global translation is a critical outcome of the integrated stress response (ISR). Consequently, it is important to effectively detect and measure protein synthesis in studies seeking to evaluate the ISR. This chapter details two methods, surface sensing of translation (SUnSET) and fluorescent noncanonical amino acid tagging (FUNCAT), to measure global translation activity in individual cells using fluorescence microscopy as a read-out. Detecting bulk translation activity in single cells is advantageous for the concurrent observation of newly synthesized proteins and other cellular structures and to identify differences in translation activity among individuals within a population of cells.


Assuntos
Biossíntese de Proteínas , Proteínas , Aminoácidos/metabolismo , Animais , Humanos , Microscopia de Fluorescência , Proteínas/metabolismo , Puromicina
7.
Stem Cell Res Ther ; 13(1): 6, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35012658

RESUMO

BACKGROUND: Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). METHODS: To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. RESULTS: The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. CONCLUSIONS: iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Citocromo P-450 CYP3A , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Puromicina/metabolismo , Puromicina/farmacologia
8.
J Mol Biol ; 434(8): 167211, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-34419431

RESUMO

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, ß-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.


Assuntos
Escherichia coli , Ribossomos , Aminoácidos/metabolismo , Dipeptídeos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Código Genético , Proteínas/metabolismo , Puromicina/metabolismo , RNA Ribossômico 23S/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
9.
Int J Mol Sci ; 22(24)2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34948207

RESUMO

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.


Assuntos
Polipose Adenomatosa do Colo/patologia , Albuminúria/patologia , Síndrome Nefrótica/patologia , Podócitos/patologia , Transcitose/fisiologia , Polipose Adenomatosa do Colo/metabolismo , Albuminúria/metabolismo , Animais , Modelos Animais de Doenças , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/metabolismo , Podócitos/metabolismo , Puromicina/farmacologia , Puromicina Aminonucleosídeo/farmacologia
10.
Biochem Biophys Res Commun ; 582: 93-99, 2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34695756

RESUMO

The genetic manipulation of cells followed by their selection is indispensable for cell biological research. Although antibiotics-resistant genes are commonly used as selection markers, optimization of the condition for each selective agent is required. Here we utilized split-inteins and the drug-selectable marker puromycin N-acetyltransferase (PAC) to develop a system that enables the selection of cells simultaneously or sequentially transfected with multiple genetic constructs, using only puromycin. The active PAC enzyme was reconstituted by intein-mediated trans-splicing at several inherent or engineered serine/cysteine residues. Multiple splitting and reconstitution of active PAC was readily achieved by selecting optimum division sites based on the cellular tolerance to various puromycin concentrations. To achieve the stepwise selection method, PAC-intein fragments were transduced into cells using a virus-like particle (VLP) composed of HIV-1 gag-pol and VSV-G. The PAC-intein-VLP successfully conferred sufficient PAC activity for puromycin selection, which was quickly diminished in the absence of the VLP. Our findings demonstrate a versatile strategy for establishing markers for all-at-once or stepwise selection of multiple genetic manipulations, which will be useful in many fields of biology.


Assuntos
Acetiltransferases/genética , Engenharia Celular/métodos , Proteínas de Fusão gag-pol/genética , Inteínas/genética , Glicoproteínas de Membrana/genética , Seleção Genética , Proteínas do Envelope Viral/genética , Acetiltransferases/metabolismo , /metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas de Fusão gag-pol/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Puromicina/farmacologia , Transfecção/métodos , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas do Envelope Viral/metabolismo
11.
Methods Mol Biol ; 2381: 267-284, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34590282

RESUMO

Genetic mutations, whether they occur within protein-coding or noncoding regions of the genome, can affect various aspects of gene expression by influencing the complex network of intra- and intermolecular interactions that occur between cellular nucleic acids and proteins. One aspect of gene expression control that can be impacted is the intracellular trafficking and translation of mRNA molecules. To study the occurrence and dynamics of translational regulation, researchers have developed approaches such as genome-wide ribosome profiling and artificial reporters that enable single molecule imaging. In this paper, we describe a complementary and optimized approach that combines puromycin labeling with a proximity ligation assay (Puro-PLA) to define sites of translation of specific mRNAs in tissues or cells. This method can be used to study the mechanisms driving the translation of select mRNAs and to access the impact of genetic mutations on local protein synthesis. This approach involves the treatment of cell or tissue specimens with puromycin to label nascently translated peptides, rapid fixation, followed by immunolabeling with appropriate primary and secondary antibodies coupled to PLA oligonucleotide probes, ligation, amplification, and signal detection via fluorescence microscopy. Puro-PLA can be performed at small scale in individual tubes or in chambered slides, or in a high-throughput setup with 96-well plate, for both in situ and in vitro experimentation.


Assuntos
Biossíntese de Proteínas , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Poliésteres , Proteínas/metabolismo , Puromicina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
12.
Methods Mol Biol ; 2320: 235-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302662

RESUMO

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) are powerful tools for elucidating the pathology behind inherited cardiomyopathies. Genome editing technologies enable targeted genome replacement and the generation of isogenic hiPSCs, allowing investigators to precisely determine the roles of identified mutations. Here, we describe a protocol to obtain isogenic hiPSCs with the corrected allele via homology-directed repair (HDR) using CRISPR/Cas9 genome editing under feeder-free conditions. Seeding hiPSCs in a 24-well plate and conducting the initial evaluation using direct genomic sequencing after 1 week is cost- and time-effective. Following optimization of the protocol, sequence confirmation of the corrected HDR clone is completed within 21 days.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Diferenciação Celular , Células Clonais/citologia , Células Clonais/metabolismo , Eletroporação/métodos , Desenho de Equipamento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Puromicina/farmacologia , Reparo de DNA por Recombinação
13.
Methods Mol Biol ; 2320: 247-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302663

RESUMO

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células Cultivadas , Células Clonais , Meios de Cultura , Primers do DNA , Resistência a Medicamentos/genética , Eletroporação , Células-Tronco Embrionárias/citologia , Edição de Genes/métodos , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Puromicina/farmacologia , RNA Guia/genética , Reparo de DNA por Recombinação/genética
14.
J Med Chem ; 64(11): 7853-7876, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34044534

RESUMO

The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.


Assuntos
Fator XIIa/antagonistas & inibidores , Peptídeos Cíclicos/química , RNA Mensageiro/metabolismo , Inibidores de Serino Proteinase/química , Sítios de Ligação , Fator XIIa/metabolismo , Biblioteca Gênica , Código Genético , Humanos , Concentração Inibidora 50 , Calicreínas/química , Calicreínas/metabolismo , Simulação de Dinâmica Molecular , Tempo de Tromboplastina Parcial , Peptídeos Cíclicos/metabolismo , Estabilidade Proteica , Tempo de Protrombina , Puromicina/química , RNA Mensageiro/química , Inibidores de Serino Proteinase/metabolismo , Relação Estrutura-Atividade
15.
J Biol Chem ; 297(1): 100839, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051232

RESUMO

Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.


Assuntos
Metabolismo Energético/genética , Glucose/farmacologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , Linhagem Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pró-Proteína Convertase 1/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
16.
BMC Microbiol ; 21(1): 120, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879049

RESUMO

BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. RESULTS: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. CONCLUSIONS: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.


Assuntos
Imipramina/farmacologia , Puromicina/análogos & derivados , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/genética , Biossíntese de Proteínas , Puromicina/química , Puromicina/metabolismo , Saccharomycetales/metabolismo , Tensoativos/farmacologia
17.
Biosci Rep ; 41(5)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33881140

RESUMO

Shensu IV is a Chinese prescription well-known for its function in treating chronic kidney diseases. However, the potential mechanisms underlying how Shensu IV exerts its effects remain unclear. In the present study, we investigated the effects of Shensu IV on glomerular podocyte injury in nephrotic rats and puromycin-induced injury in cultured podocytes, and assessed the associated molecular mechanisms. Liquid chromatography-mass spectrometry (LC-MS) results showed that the main components of Shensu IV were l-Carnitine, P-lysoPC (LPC) 16:0, Coumaroyl tyramine, Tetramethylpyrazine, LPC 18:1, Choline, (S,S)-Butane-2,3-diol, and Scopoletin. We further found that nephrotic rats displayed pathological alterations in kidney tissues and ultrastructural changes in glomerular podocytes; however, these effects were reversed with Shensu IV treatment. Compared with the control, the numbers of autophagosomes were markedly reduced in the model group, but not in the Shensu IV treatment group. Furthermore, the expression of p62 was significantly higher in the model group than in the controls, whereas the LC3-II/I ratio was significantly lower; however, these changes were not observed when Shensu IV was administered. The protective effects of Shensu IV were further confirmed in podocytes displaying puromycin-induced injury. Compared with control group, the expression of long non-coding RNA (lncRNA) H19, mTOR, p-mTOR, and p62 was significantly increased in the puromycin group, whereas that of distinct subgroup of the RAS family member 3 (DIRAS3) was significantly decreased, as was the LC3-II/I ratio. The opposite results were obtained for both shH19- and Shensu IV-treated cells. Collectively, our data demonstrated that Shensu IV can prevent glomerular podocyte injury in nephrotic rats and puromycin-treated podocytes, likely via promoting lncRNA H19/DIRAS3-regulated autophagy.


Assuntos
Autofagia , Medicamentos de Ervas Chinesas/uso terapêutico , Nefrose/tratamento farmacológico , Podócitos/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , Proteínas rho de Ligação ao GTP/genética , Animais , Células Cultivadas , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Nefrose/etiologia , Nefrose/prevenção & controle , Podócitos/metabolismo , Puromicina/toxicidade , RNA Longo não Codificante/genética , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
18.
Anal Chem ; 93(16): 6403-6413, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33856767

RESUMO

Nascent proteome is crucial in directly revealing how the expression of a gene is regulated on a translation level. In the nascent protein identification, puromycin capture is one of the pivotal methods, but it is still facing the challenge in the deep profiling of nascent proteomes due to the low abundance of most nascent proteins. Here, we describe the synthesis of puromycin-modified silica microspheres (PMSs) as the sorbent of dispersive solid-phase microextraction and the establishment of the PMS-based nascent proteomics (PMSNP) method for efficient capture and analysis of nascent proteins. The modification efficiency of puromycin groups on silica microspheres reached 91.8% through the click reaction. After the optimization and simplification of PMSNP, more than 3500 and 3900 nascent proteins were rapidly identified in HeLa cells and mouse brains within 13.5 h, respectively. The PMSNP method was successfully applied to explore changes in the translation process in a biological stress model, namely, the lipopolysaccharide-stimulated HeLa cells. Biological functional analyses revealed the unique characters of the nascent proteomes and exhibited the superiority of the PMSNP in the identification of low abundance and secreted nascent proteins, thus demonstrating the sensitivity and immediacy of the PMSNP method.


Assuntos
Microesferas , Proteoma , Proteômica , Puromicina , Células HeLa , Humanos , Proteoma/análise , Dióxido de Silício
19.
FASEB J ; 35(4): e21444, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33749901

RESUMO

Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are the classical regulator of the hypoxia inducible factor (HIF) signal pathway, many researchers found that PHDs are involved in the muscle fiber type transformation, muscle regeneration, and myocyte differentiation. However, whether PHDs can impact the protein turnover of skeletal muscle is poorly understood. In this study, we constructed denervated muscle atrophy mouse model and found PHD3 was highly expressed in the atrophic muscles and there was a significant correlation between the expression level of PHD3 and skeletal muscle weight which was distinct from PHD1 and PHD2. Then, the similar results were getting from the different weight muscles of normal mice. To further verify the relationship between PHD3 and skeletal muscle protein turnover, we established a PHD3 interference model by injecting PHD3 sgRNA virus into tibialis anterior muscle (TA) muscle of MCK-Cre-cas9 mice and transfecting PHD3 shRNA lentivirus into primary satellite cells. It was found that the Knock-out of PHD3 in vivo led to a significant increase in muscle weight and muscle fiber area (P < .05). Besides, the activity of protein synthesis signal pathway increased significantly, while the protein degradation pathway was inhibited evidently (P < .05). In vitro, the results of 5-ethynyl-2'-deoxyuridine (EdU) and tetramethylrhodamine ethyl ester (TMRE) fluorescence detection showed that PHD3 interference could lead to a decrease in cell proliferation and an increase of cell apoptosis. After the differentiation of satellite cells, the production of puromycin in the interference group was higher than that in the control group, and the content of 3-methylhistidine in the interference group was lower than that in the control group (P < .05) which is consistent with the change of protein turnover signal pathway in the cell. Mechanistically, there is an interaction between PHD3, NF-κB, and IKBα which was detected by immunoprecipitation. With the interfering of PHD3, the expression of the inflammatory signal pathway also significantly decreased (P < .05). These results suggest that PHD3 may affect protein turnover in muscle tissue by mediating inflammatory signal pathway. Finally, we knocked out PHD3 in denervated muscle atrophy mice and LPS-induced myotubes atrophy model. Then, we found that the decrease of PHD3 protein level could alleviate the muscle weight and muscle fiber reduction induced by denervation in mice. Meanwhile, the protein level of the inflammatory signal pathway and the content of 3-methylhistidine in denervated atrophic muscle were also significantly reduced (P < .05). In vitro, PHD3 knock-out could alleviate the decrease of myotube diameter induced by LPS, and the expression of protein synthesis pathway was also significantly increased (P < .05). On the contrary, the expression level of protein degradation and inflammatory signal pathway was significantly decreased (P < .05). Through these series of studies, we found that the increased expression of PHD3 in denervated muscle might be an important regulator in inducing muscle atrophy, and this process is likely to be mediated by the inflammatory NF-κB signal pathway.


Assuntos
Denervação , Músculo Esquelético/inervação , Atrofia Muscular/metabolismo , NF-kappa B/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Regulação da Expressão Gênica , Hipertrofia , Inflamação/genética , Inflamação/metabolismo , Metilistidinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , NF-kappa B/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Puromicina , Células Satélites de Músculo Esquelético/fisiologia , Transdução de Sinais
20.
Sci Rep ; 11(1): 5247, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664348

RESUMO

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.


Assuntos
Acetilcoenzima A/química , Acetiltransferases/ultraestrutura , Proteínas Recombinantes/ultraestrutura , Streptomyces/ultraestrutura , Acetilcoenzima A/genética , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Animais , Domínio Catalítico/genética , Linhagem Celular , Cristalografia por Raios X , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mutação/genética , Puromicina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Streptomyces/enzimologia
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