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1.
PLoS Genet ; 16(10): e1009068, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33057331

RESUMO

Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.


Assuntos
Quinase do Fator 2 de Elongação/genética , Histidina/análogos & derivados , Fatores de Transcrição Kruppel-Like/genética , Antígenos de Histocompatibilidade Menor/genética , Proteínas Supressoras de Tumor/genética , Animais , Domínio BTB-POZ/genética , Sistemas CRISPR-Cas/genética , Regulação da Expressão Gênica/genética , Histidina/biossíntese , Histidina/genética , Humanos , Metiltransferases , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Células RAW 264.7 , Fatores de Transcrição/genética
2.
Nat Med ; 26(10): 1602-1608, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32747827

RESUMO

Artemisinin resistance (delayed P. falciparum clearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa1-4. Here we genotyped the P. falciparum K13 (Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance5,6, in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda7. While cure rates were >95% in both treatment arms, the Pfkelch13 R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.


Assuntos
Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Mutação de Sentido Incorreto , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Substituição de Aminoácidos/genética , Animais , Arginina/genética , Evolução Clonal/genética , Doenças Transmissíveis Emergentes/tratamento farmacológico , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/parasitologia , Genótipo , Histidina/genética , Humanos , Técnicas In Vitro , Repetição Kelch/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Testes de Sensibilidade Parasitária , Filogenia , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo Genético , Proteínas de Protozoários/química , Ruanda/epidemiologia
3.
J Chromatogr A ; 1629: 461505, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32861092

RESUMO

Immobilized metal affinity chromatography (IMAC) is a technique primarily used in research and development laboratories to purify proteins containing engineered histidine tags. Although this type of chromatography is commonly used, it can be problematic as differing combinations of resins and metal chelators can result in highly variable chromatographic performance and product quality results. To generate a robust IMAC purification process, the binding differences of resin and metal chelator combinations were studied by generating breakthrough curves with a poly-histidine tagged bispecific protein. The optimal binding combination was statistically analyzed to determine the impact of chromatographic parameters on the operation. Additionally, equilibrium uptake isotherms were created to further elucidate the impact of chromatographic parameters on the binding of protein. It was found that for protein expressed in CHO cells, Millipore Sigma's Fractogel EMD Chelate (M) charged with Zn2+ and GE's pre-charged Ni Sepharose Excel displayed the highest binding capacities. When the protein was expressed in HEK-293, GE's IMAC Sepharose 6 Fast Flow charged with either Co2+ or Zn2+ bound the greatest amount of protein. The study further identified the metal binding capacity of the resin lot, the protein capacity to which the resin is loaded, and the ratio of poly-histidine tag residues on the protein all impacted the chromatographic performance and product quality. These findings enabled the development of a robust and scalable process. The CHO expressed cell culture product was directly loaded at a high capacity onto variable metal binding affinity Fractogel EMD Chelate (M). A 250 mM imidazole elution condition ensured the product contained monomeric 4 and 6-histidine tagged bispecific proteins. The optimized IMAC process conditions determined in this study can be applied to a wide variety of poly-histidine tagged proteins in research and development laboratories as various poly-histidine tagged proteins of differing molecular weights and formats expressed in either HEK-293 or CHO cells were successfully purified.


Assuntos
Cromatografia de Afinidade/métodos , Histidina/metabolismo , Metais/química , Proteínas Recombinantes/isolamento & purificação , Animais , Células CHO , Quelantes/química , Cromatografia de Fase Reversa , Cobalto/química , Cricetinae , Cricetulus , Células HEK293 , Histidina/genética , Humanos , Proteínas Recombinantes/biossíntese , Zinco/química
4.
Arch Virol ; 165(10): 2301-2309, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757056

RESUMO

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.


Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Bombyx/virologia , Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Baculoviridae/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/imunologia , Soros Imunes/química , Imunogenicidade da Vacina , Larva/virologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
5.
Exp Cell Res ; 391(1): 111987, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32240661

RESUMO

BACKGROUND: The protein plasminogen activator inhibitor-1 (PAI-1), an inhibitor specific for urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA), has been shown to have a key role in cancer metastases. Currently, it is unknown as to whether the exocellular inhibition of PAI-1 can inhibit the migration of cancer cells. METHODS: By fusing the mutated serine protease domain (SPD) of uPA and human serum albumin (HSA), PAItrap3, a protein that traps PAI-1, was synthesized and experiments were conducted to determine if exocellular PAItrap3 attenuates PAI-1-induced cancer cell migration in vitro. RESULTS: PAItrap3 (0.8 µM) significantly inhibited the motility of MCF-7, MDA-MB-231, HeLa and 4T1 cancer cells, by 90%, 50%, 30% and 20%, respectively, without significantly altering their proliferation. The PAI-1-induced rearrangement of F-actin was significantly inhibited by PAItrap3, which produced a decrease in the number of cell protrusions by at least 20%. CONCLUSIONS: In vitro, PAItrap3 inhibited PAI-1-induced cancer cell migration, mainly through inhibiting the rearrangement of F-actin. Overall, these results, provided they can be extrapolated to humans, suggest that the PAItrap3 protein could be used as an exocellular inhibitor to attenuate cancer metastases.


Assuntos
Actinas/genética , Movimento Celular/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Inibidor da Proteína C/farmacologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Sítios de Ligação , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Células MCF-7 , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Pichia/genética , Pichia/metabolismo , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Inibidor da Proteína C/química , Inibidor da Proteína C/genética , Inibidor da Proteína C/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Nat Commun ; 11(1): 1757, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273505

RESUMO

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical.


Assuntos
Proteínas de Bactérias/metabolismo , Methanosarcina/metabolismo , Nitrogênio/metabolismo , Nitrogenase/biossíntese , Alanina/genética , Alanina/metabolismo , Proteínas de Bactérias/genética , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/genética , Histidina/metabolismo , Ligantes , Methanosarcina/genética , Mutagênese , Nitrogenase/genética , Espectroscopia por Absorção de Raios X
7.
PLoS One ; 15(3): e0230282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160243

RESUMO

Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexa-histidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.


Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Mycobacterium/genética , Escherichia coli/genética , Genes Reporter , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional
8.
Biochim Biophys Acta Biomembr ; 1862(7): 183236, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126226

RESUMO

Piscidins are host-defense peptides (HDPs) from fish that exhibit antimicrobial, antiviral, anti-cancer, anti-inflammatory, and wound-healing properties. They are distinctively rich in histidine and contain an amino terminal copper and nickel (ATCUN) binding motif due to the presence of a conserved histidine at position 3. Metallation lowers their total charge and provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs). Here, we focus on P1, a particularly membrane-active isoform, and investigate how metallating it and making OxPL available influence its membrane activity. First, we quantify through dye leakage experiments the permeabilization of the apo- and holo-forms of P1 on model membranes containing a fixed ratio of anionic phosphatidylglycerol (PG) and zwitterionic phosphatidylcholine (PC) but varying amounts of Aldo-PC, an OxPL derived from the degradation of several UFAs. Remarkably, metallating P1 increases membranolysis by a factor of five in each lipid system. Conversely, making Aldo-PC available improves permeabilization by a factor of two for each peptide form. Second, we demonstrate through CD-monitored titrations that the strength of the peptide-membrane interactions is similar in PC/PG and PC/PG/Aldo-PC. Thus, peptide-induced membrane activity is boosted by properties intrinsic to the peptide (e.g., charge and structural changes associated with metallation) and bilayer (e.g., reversal of sn-2 chain due to oxidation). Third, we show using oriented-sample 15N solid-state NMR that the helical portion of P1 lies parallel to the bilayer surface in both lipid systems. 31P NMR experiments show that both the apo- and holo-states interact more readily with PC in PC/PG. However, the presence of Aldo-PC renders the holo-, but not the apo-state, more specific to PG. Hence, the membrane disruptive effects of P1 and its specificity for the anionic lipids found on pathogenic cell membrane surfaces are simultaneously optimized when it is metallated and the OxPL is present. Overall, this study deepens our insights into how OxPLs affect peptide-lipid interactions and how host defense metallopeptides could help integrate the effects of antimicrobial agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Ácidos Graxos Insaturados/química , Proteínas de Peixes/genética , Metais/química , Animais , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sítios de Ligação , Membrana Celular , Cobre/química , Ácidos Graxos Insaturados/genética , Proteínas de Peixes/química , Histidina/química , Histidina/genética , Humanos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Níquel/química , Fosfolipídeos/química , Fosfolipídeos/genética
9.
Science ; 367(6479): 806-810, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32001525

RESUMO

Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We used single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near-atomic resolution. Glutamine-148→histidine (Q148H) and glycine-140→serine (G140S) amino acid substitutions in integrase that result in clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone that are critical for antagonizing viruses containing the Q148H and G140S mutations. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this class of anti-HIV/AIDS therapeutics.


Assuntos
Farmacorresistência Viral , Inibidores de Integrase de HIV/química , Integrase de HIV/química , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Substituição de Aminoácidos/genética , Domínio Catalítico , Microscopia Crioeletrônica/métodos , Glutamina/genética , Glicina/genética , Integrase de HIV/genética , Inibidores de Integrase de HIV/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histidina/genética , Humanos , Magnésio/química , Mutação , Oxazinas , Piperazinas , Piridonas , Serina/genética , Imagem Individual de Molécula/métodos
10.
Protein Expr Purif ; 170: 105593, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32032772

RESUMO

Cost-effectiveness is an important issue in biotechnological manufacturing industry and using alternative cheap materials with the same benefits has been noticed in most literatures. Isopropyl ß-d-1-thiogalactopyranoside (IPTG), a well-known chemical element for induction of protein expression, has several disadvantages such as high expense and toxicity. In this study, we aimed to introduce skimmed milk as an alternative material for protein expression by induction of lac operon. In this way, Escherichia coli BL21 (DE3) bacteria were induced using 1 mM IPTG or 1.0% (w/v) skimmed milk. Protein purification was performed using Ni-NTA (nickel-nitrilotriacetic acid) for His-tagged recombinant proteins and protein purity was evaluated by SDS-PAGE. Results showed high level of recombinant protein expression using skimmed milk, and interestingly, the growth rate of bacteria improved. Our findings suggested that skimmed milk can be a suitable alternative for induction of recombinant protein expression, which has advantages such as more availability and affordability, in comparison to IPTG supplementation.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Flagelina/genética , Lactose/farmacologia , Leite/química , Proteínas Recombinantes de Fusão/genética , Animais , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelina/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Isopropiltiogalactosídeo/farmacologia , Óperon Lac/efeitos dos fármacos , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/química
11.
Protein Expr Purif ; 170: 105607, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32062022

RESUMO

It is well known that camelids (camels and llamas) have fully functional antibodies with only a heavy chain consisting of a single variable domain and two constant domains. This single variable domain is called a "nanobody" and many nanobodies are synthesized in the cytosol of Escherichia coli, however, most of the nanobodies become inclusion bodies without tags to enhance their solubility. We generated a vector system to enable the secretary expression of nanobodies in Escherichia coli. In this system, several NBs were secreted into the culture supernatant. Since the vector contained 6xHis tag and AviTAG, biotinylation (even fluorescent-labeled) of AviTAG was achieved during cell culture, and purification of the supernatant was a step by immobilized metal ion adsorption chromatography. The procedure described in this study is believed to be as simple as regular plasmid minipreps. Therefore, many laboratories can use this method.


Assuntos
Escherichia coli/metabolismo , Plasmídeos/metabolismo , Anticorpos de Domínio Único/isolamento & purificação , Animais , Avidina/química , Biotinilação , Camelídeos Americanos , Camelus , Cromatografia de Afinidade , Clonagem Molecular , Meios de Cultivo Condicionados/química , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Plasmídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo
12.
Biochim Biophys Acta Biomembr ; 1862(8): 183212, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057757

RESUMO

The LAH4 family of amphipathic peptides exhibits pronounced antimicrobial, cell penetrating and nucleic acid transfection activities. Furthermore, variants were designed with potent lentiviral transduction enhancement. When viewed along a helical wheel the four histidines are arranged to form an amphipathic structure. In order to optimize some of these biological activities the number of leucine and alanine residues exposed to the hydrophilic surface was systematically varied which resulted in the design of vectofusin a peptide with strong lentiviral transduction enhancement activities. Here the series of peptides with varying numbers of alanine or leucine residues, respectively, framed by the histidines was tested for their calcein release activity. Interestingly, the membrane pore formation and DNA transfection activities show a clear correlation with the hydrophilic angle. In contrast the membrane partitioning and the propensity to adopt helical conformations was hardly affected as long as the hydrophilic angle did not exceed a limiting value of 150°.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , DNA/genética , Histidina/genética , Membranas/efeitos dos fármacos , Alanina/genética , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , DNA/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lentivirus/genética , Leucina/genética , Membranas/metabolismo , Porosidade , Transfecção
13.
Plant Sci ; 293: 110407, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32081257

RESUMO

Leaf color mutants are an ideal tool to study chlorophyll biosynthesis, chloroplast development and photosynthesis. In this study, we identified an EMS-induced yellow young leaf mutant C777. The mutant exhibited yellow cotyledons and emerging true leaves with stay-green dots that turn green gradually with leaf growth. Segregation analysis in several populations indicated that the mutant C777 was controlled by a recessive gene yyl-1. Fine mapping delimited the yyl-1 locus to a 45.3 kb region harboring 8 putative genes, but only one SNP (G to A) was identified between C777 and its wild-type parental line in this region which occurred in the 13th exon of CsHD that encodes a histidine and aspartic acid (HD) domain containing protein. This nonsense mutation introduced a stop codon and thus a premature protein. Uniqueness of this mutant allele was verified in 515 cucumber lines. Quantitative real-time PCR revealed significantly reduced expression of CsHD gene in the mutant. Further, silencing the NbHD gene by VIGS in tobacco resulted in virescent young leaves and significantly down-regulated expression of HD gene. These results strongly supported the association of the CsHD gene with the virescent young leaf phenotype in C777. This is the first report to clone and characterize the CsHD gene in the horticultural crops. The results may help understand the functions of the HD gene in chloroplast development and chlorophyll biosynthesis in plants.


Assuntos
Ácido Aspártico/genética , Cucumis sativus/genética , Genes de Plantas/genética , Histidina/genética , Mutação , Proteínas de Plantas/genética , Clorofila/biossíntese , Clorofila/genética , Cloroplastos/genética , Mapeamento Cromossômico , Clonagem Molecular , Cor , DNA de Plantas/genética , Éxons , Regulação da Expressão Gênica de Plantas , Genes Recessivos , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Domínios Proteicos , Tabaco
14.
Anal Chem ; 92(5): 3913-3922, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-31992042

RESUMO

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.


Assuntos
Cromatografia de Afinidade/métodos , Mapas de Interação de Proteínas/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Cromatografia Líquida de Alta Pressão , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Ácido Nitrilotriacético/química , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/análise , Ligação Proteica , Proteômica/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas em Tandem
15.
Curr Microbiol ; 77(5): 710-715, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31897665

RESUMO

Phosphatidylserine synthase (Pss) is involved in the metabolic pathway in phospholipid synthesis in different organisms. In this study, Pss expression in Vibrio parahaemolyticus was verified through liquid chromatography tandem-mass spectrometry. To analyze the characteristics of Pss, the recombinant Pss was overexpressed and purified from Escherichia coli. The optimum temperature and pH of Pss were 40 °C and 8, respectively. When reacting with divalent metal, Pss activity decreased. In addition, Pss could not only use cytidine diphosphate diacylglycerol (CDP-DAG, 16:0), but also CDP-DAG (18:1) as a substrate to produce cytidine 5'-monophosphate. Furthermore, the 3D structure of Pss was predicted, and the results revealed that histidine and lysine of the two HKD motifs were present in the catalytic site. Moreover, CDP-DAG (16:0) was docked with the Pss model. To investigate whether the two HKD motifs in Pss are important to its activity, site-directed mutagenesis of histidine was performed. The results revealed that the activities of both H131A and H352A were diminished. Little is known regarding the catalytic site of type I Pss. This is the first report on the biochemical characterization of Pss in V. parahaemolyticus.


Assuntos
Proteínas de Bactérias/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Vibrio parahaemolyticus/enzimologia , Proteínas de Bactérias/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Cromatografia Líquida , Escherichia coli/genética , Histidina/genética , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Fosfolipídeos/metabolismo , Espectrometria de Massas em Tandem , Temperatura , Vibrio parahaemolyticus/genética
16.
Int J Mol Sci ; 21(3)2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31979156

RESUMO

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.


Assuntos
Proteínas de Bactérias/genética , Geobacillus stearothermophilus/genética , Histidina/genética , Mutação/genética , Fatores de Iniciação de Peptídeos/genética , Substituição de Aminoácidos/genética , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/genética , Fenótipo , Biossíntese de Proteínas/genética , Domínios Proteicos/genética , RNA de Transferência de Metionina/genética , Ribossomos/genética
18.
J Steroid Biochem Mol Biol ; 196: 105507, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31669572

RESUMO

Aromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, reduce CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, the effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C, and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284 L as well as the POR-P284 T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained approximately 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing toward a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point toward a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284 L, P284 T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Aromatase/genética , Aromatase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hiperplasia Suprarrenal Congênita/enzimologia , Hiperplasia Suprarrenal Congênita/metabolismo , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Androstenodiona/metabolismo , Arginina/genética , Aromatase/química , Aromatase/deficiência , Cisteína/genética , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/deficiência , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Ativação Enzimática/genética , Histidina/genética , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto/fisiologia , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Relação Estrutura-Atividade
19.
Genes Cells ; 25(2): 76-85, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31828897

RESUMO

Eukaryotic elongation factor 2 (eEF2) undergoes a unique post-translational modification called diphthamidation. Although eEF2 diphthamidation is highly conserved, its pathophysiological function is still largely unknown. To elucidate the function of diphthamidation in tumor, we examined the involvement of diphthamidation pathway enzyme Dph5 in tumor progression in Drosophila adult gut. Expression of oncogenic RasV12 in gut intestinal stem cells (ISCs) and enteroblasts (EBs) causes hypertrophy and disruption of gut epithelia, and shortened life span. Knockdown of Dph5 ameliorated these pathogenic phenotypes. Dph5 is required for gross translation activation and high dMyc protein level in RasV12 tumor-like hyperplasia. Transcriptome analysis revealed that Dph5 is involved in the regulation of ribosome biogenesis genes. These results suggest that diphthamidation is required for translation activation partly through the regulation of ribosome biogenesis in Ras-induced tumor-like hyperplasia model in Drosophila gut.


Assuntos
Neoplasias Gastrointestinais/metabolismo , Genes ras/genética , Histidina/genética , Histidina/metabolismo , Hiperplasia/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Animais , Drosophila/genética , Ingestão de Alimentos , Eucariotos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histidina/análogos & derivados , Masculino , Processamento de Proteína Pós-Traducional , Ribossomos/genética , Transcriptoma
20.
MAbs ; 12(1): 1682866, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31777319

RESUMO

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.


Assuntos
Antineoplásicos Imunológicos/química , Imunoterapia/métodos , Neoplasias/terapia , Afinidade de Anticorpos/genética , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral , Histidina/genética , Humanos , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Neoplasias/imunologia , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Receptor ErbB-2/imunologia , Trastuzumab/uso terapêutico , Microambiente Tumoral
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