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World J Microbiol Biotechnol ; 38(12): 252, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36316502

RESUMO

Chitinases are a group of enzymes that catalyze chitin hydrolysis and are present in all domains of life. Chitinases belong to different glycosyl hydrolase families with great diversity in their sequences. Microorganisms such as bacteria and fungi produce chitinases for nutrition, and energy, and to parasitize the chitinous hosts. But chitinases from bacteria are of special interest due to their ubiquitous nature and ability to perform under extreme conditions. Chitinases produced by bacteria have been explored for their use in agriculture and industry. In agriculture, their main role is to control chitin-containing insect pests, fungal pathogens, and nematodes. In the seafood industry, they found their role in the management of processing wastes which are mainly chitinous substances. Chitinases are also used to synthesize low molecular weight chitooligomers which are proven bioactive compounds with activities such as anti-tumour, antimicrobial, and immunity modulation. Considering their importance in ecology and biotechnological applications, several bacterial chitinases have been studied in the last two decades. Despite their potential, bacterial chitinases have a few limitations such as low production and lack of secretion systems which make the wild-type enzymes unfit for their applications in industries and other allied sectors. This review is an attempt to collate significant works in bacterial chitinases and their application in various industries and the employment of various tools and techniques for improvement to meet industrial requirements.


Assuntos
Bactérias , Quitinases , Bactérias/enzimologia , Biotecnologia/métodos , Quitina , Quitinases/biossíntese , Hidrólise
3.
Science ; 378(6618): 405-412, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36302022

RESUMO

To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5S, 16S, and 23S ribosomal RNAs from enzymatically assembled long mirror-image genes. We further exploited the versatile mirror-image T7 transcription system for practical applications such as biostable mirror-image riboswitch sensor, long-term storage of unprotected kilobase-long l-RNA in water, and l-ribozyme-catalyzed l-RNA polymerization to serve as a model system for basic RNA research.


Assuntos
DNA Polimerase Dirigida por DNA , RNA Catalítico , RNA Ribossômico 23S , RNA Ribossômico 5S , Ribossomos , Transcrição Genética , Proteínas Virais , Conformação de Ácido Nucleico , RNA Catalítico/genética , RNA Ribossômico 23S/biossíntese , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 5S/biossíntese , RNA Ribossômico 5S/genética , DNA Polimerase Dirigida por DNA/síntese química , DNA Polimerase Dirigida por DNA/química , Proteínas Virais/síntese química , Proteínas Virais/química , RNA Ribossômico 16S/biossíntese , RNA Ribossômico 16S/genética
4.
Int J Mol Sci ; 23(19)2022 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-36232994

RESUMO

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.


Assuntos
Paracoccus , Saccharomycetales , beta-Galactosidase , Fermentação , Galactosidases , Paracoccus/enzimologia , Saccharomycetales/metabolismo , beta-Galactosidase/biossíntese
5.
Chem Commun (Camb) ; 58(87): 12139-12150, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36222364

RESUMO

The biosynthesis of glycans is a highly conserved biological process and found in all domains of life. The expression of cell surface glycans is increasingly recognized as a target for therapeutic intervention given the role of glycans in major pathologies such as cancer and microbial infection. Herein, we summarize our contributions to the development of unnatural monosaccharide derivatives to infiltrate and alter the expression of both mammalian and bacterial glycans and their therapeutic application.


Assuntos
Fucose , Monossacarídeos , Polissacarídeos , Animais , Fucose/química , Mamíferos , Monossacarídeos/química , Ácido N-Acetilneuramínico/química , Polissacarídeos/biossíntese , Polissacarídeos/química , Bactérias
6.
Appl Microbiol Biotechnol ; 106(23): 7805-7817, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36260100

RESUMO

Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein (pal) gene. Ulp1403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δpal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1403-621 approximately four fold. Moreover, extracellular Ulp1403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus. The specific activity of extracellular Ulp1403-621 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 106 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. KEY POINTS: • First report of extracellular production of Ulp1403-621 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1403-621 approximately four fold. • The specific activity of extracellular Ulp1403-621 produced in this study reached 2.0 × 106 U/L.


Assuntos
Peptídeos Antimicrobianos , Escherichia coli , Proteínas Recombinantes , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/metabolismo , Glicina/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Ubiquitinas/metabolismo
7.
Appl Microbiol Biotechnol ; 106(21): 7063-7072, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36195703

RESUMO

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.


Assuntos
Proteínas Fúngicas , Pró-Proteína Convertases , Saccharomyces cerevisiae , Candida albicans/enzimologia , Candida albicans/genética , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Pró-Proteína Convertases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo , Proteínas Fúngicas/biossíntese
8.
Nature ; 610(7932): 562-568, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36261549

RESUMO

Tobacco smoking is positively correlated with non-alcoholic fatty liver disease (NAFLD)1-5, but the underlying mechanism for this association is unclear. Here we report that nicotine accumulates in the intestine during tobacco smoking and activates intestinal AMPKα. We identify the gut bacterium Bacteroides xylanisolvens as an effective nicotine degrader. Colonization of B. xylanisolvens reduces intestinal nicotine concentrations in nicotine-exposed mice, and it improves nicotine-exacerbated NAFLD progression. Mechanistically, AMPKα promotes the phosphorylation of sphingomyelin phosphodiesterase 3 (SMPD3), stabilizing the latter and therefore increasing intestinal ceramide formation, which contributes to NAFLD progression to non-alcoholic steatohepatitis (NASH). Our results establish a role for intestinal nicotine accumulation in NAFLD progression and reveal an endogenous bacterium in the human intestine with the ability to metabolize nicotine. These findings suggest a possible route to reduce tobacco smoking-exacerbated NAFLD progression.


Assuntos
Bactérias , Intestinos , Nicotina , Hepatopatia Gordurosa não Alcoólica , Fumar Tabaco , Animais , Humanos , Camundongos , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Ceramidas/biossíntese , Nicotina/efeitos adversos , Nicotina/metabolismo , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/microbiologia , Esfingomielina Fosfodiesterase/metabolismo , Fumar Tabaco/efeitos adversos , Fumar Tabaco/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Progressão da Doença
9.
Front Endocrinol (Lausanne) ; 13: 1005639, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36299464

RESUMO

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.


Assuntos
Ceramidas , Vesículas Extracelulares , Rim Policístico Autossômico Dominante , Humanos , Trifosfato de Adenosina , Apirase/metabolismo , Ceramidas/biossíntese , Ceramidas/genética , Vesículas Extracelulares/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética
10.
Nature ; 610(7933): 783-790, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36224385

RESUMO

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to ß-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2ß2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and ß-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α-HIF1ß heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for ß-haemoglobinopathies.


Assuntos
gama-Globinas , Humanos , Cromatina , Hemoglobina Fetal/biossíntese , Hemoglobina Fetal/genética , gama-Globinas/biossíntese , gama-Globinas/genética , Hipóxia/genética , Prolil Hidroxilases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Longo não Codificante , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Eritropoese
11.
PLoS One ; 17(10): e0276775, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36301906

RESUMO

Plasma fibrinogen and albumin concentrations initially decrease after abdominal surgery. On postoperative days 3-5 fibrinogen concentration returns to the preoperative level or even higher, while albumin stays low. It is not known if these altered plasma concentrations reflect changes in synthesis rate, utilization, or both. In particular a low albumin plasma concentration has often been attributed to a low synthesis rate, which is not always the case. The objective of this study was to determine fibrinogen and albumin quantitative synthesis rates in patients undergoing major upper abdominal surgery with and without intact liver size. Patients undergoing liver or pancreatic resection (n = 9+6) were studied preoperatively, on postoperative days 1 and 3-5. De novo synthesis of fibrinogen and albumin was determined; in addition, several biomarkers indicative of fibrinogen utilization were monitored. After hemihepatectomy, fibrinogen synthesis was 2-3-fold higher on postoperative day 1 than preoperatively. On postoperative days 3-5 the synthesis level was still higher than preoperatively. Following major liver resections albumin synthesis was not altered postoperatively compared to preoperative values. After pancreatic resection, on postoperative day 1 fibrinogen synthesis was 5-6-fold higher than preoperatively and albumin synthesis 1.5-fold higher. On postoperative days 3-5, synthesis levels returned to preoperative levels. Despite decreases in plasma concentrations, de novo synthesis of fibrinogen was markedly stimulated on postoperative day 1 after both hemihepatectomies and pancreatectomies, while de novo albumin synthesis remained grossly unchanged. The less pronounced changes seen following hepatectomies were possibly related to the loss of liver tissue.


Assuntos
Procedimentos Cirúrgicos do Sistema Digestório , Fibrinogênio , Hemostáticos , Albumina Sérica , Humanos , Abdome/cirurgia , Fibrinogênio/biossíntese , Hepatectomia , Fígado/cirurgia , Albumina Sérica/biossíntese
12.
Nature ; 609(7929): 1038-1047, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36171374

RESUMO

Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.


Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , Reparo do DNA , Estresse Oxidativo , Regiões Promotoras Genéticas , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Genes , Teste de Complementação Genética , Mitose , Mutação , Estresse Oxidativo/genética , Diester Fosfórico Hidrolases/metabolismo , Poli ADP Ribosilação , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA/genética , RNA Polimerase II/metabolismo , Fuso Acromático/metabolismo , Sítio de Iniciação de Transcrição
13.
J Nat Prod ; 85(9): 2236-2250, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36098709

RESUMO

This Review provides a critical analysis of the literature covering the naturally occurring partially reduced perylenequinones (PQs) from fungi without carbon substituents (which can be named class A perylenequinones) and discusses their structures, stereochemistry, biosynthesis, and biological activities as appropriate. Perylenequinones are natural pigments with a perylene skeleton produced by certain fungi, aphids, some plants, and animal species. These compounds display several biological activities, e.g., antimicrobial, anti-HIV, photosensitizers, cytotoxic, and phytotoxic. It describes 36 fungal PQs and cites 81 references, covering from 1956 to August 2022.


Assuntos
Fungos , Perileno , Pigmentos Biológicos , Quinonas , Animais , Fungos/química , Perileno/análogos & derivados , Perileno/química , Perileno/farmacologia , Fármacos Fotossensibilizantes , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/química , Pigmentos Biológicos/isolamento & purificação , Pigmentos Biológicos/farmacologia , Quinonas/química , Quinonas/farmacologia
14.
Semin Cancer Biol ; 86(Pt 2): 93-100, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36096316

RESUMO

The energy metabolism of tumor cells is considered one of the hallmarks of cancer because it is different from normal cells and mainly consists of aerobic glycolysis, fatty acid oxidation, and glutaminolysis. It is about one hundred years ago since Warburg observed that cancer cells prefer aerobic glycolysis even in normoxic conditions, favoring their high proliferation rate. A pivotal enzyme driving this phenomenon is lactate dehydrogenase (LDH), and this review describes prognostic and therapeutic opportunities associated with this enzyme, focussing on tumors with limited therapeutic strategies and life expectancy (i.e., pancreatic and thoracic cancers). Expression levels of LDH-A in pancreatic cancer tissues correlate with clinicopathological features: LDH-A is overexpressed during pancreatic carcinogenesis and showed significantly higher expression in more aggressive tumors. Similarly, LDH levels are a marker of negative prognosis in patients with both adenocarcinoma or squamous cell lung carcinoma, as well as in malignant pleural mesothelioma. Additionally, serum LDH levels may play a key role in the clinical management of these diseases because they are associated with tissue damage induced by tumor burden. Lastly, we discuss the promising results of strategies targeting LDH as a treatment strategy, reporting recent preclinical and translational studies supporting the use of LDH-inhibitors in combinations with current/novel chemotherapeutics that can synergistically target the oxygenated cells present in the tumor.


Assuntos
Metabolismo Energético , Lactato Desidrogenase 5 , Neoplasias Pancreáticas , Neoplasias Torácicas , Humanos , Glicólise/fisiologia , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5/biossíntese , Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Neoplasias Torácicas/metabolismo
15.
BMC Cancer ; 22(1): 990, 2022 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-36115941

RESUMO

BACKGROUND: A group of genetically altered cells that have not transformed into a clinical or histologically identifiable state of malignancy but contains a higher risk of transforming into one is known as the field of cancerization. Numerous molecules are being investigated for their significance in the development of this phenomenon. One such protein of this family is Kaiso also known as ZBTB33 (Zinc Finger and BTB Domain containing 33). This protein belongs to the POZ-ZF family of transcription factors and may have functional tasks similar to its other siblings such as the growth and development of vertebrates and the pathogenesis of neoplastic diseases. Nevertheless, its role in the pathogenesis, progression, epithelial mesenchyal transition and field cancerization in case of oral cancer still needs exploration. Hence, this study was designed to explore the expressional differences between the mucosa of controls and those diagnosed with oral squamous cell carcinoma (OSCC). METHODS: Soft tissue samples were obtained from the main tumor, tumor periphery and opposite buccal mucosa of 50 oral cancer patients, whereas normal mucosa was taken from 50 volunteers undergoing elective tooth removal. The acquired samples were subjected to Immunohistochemical exploration for expression of Kaiso and E-Cadherin. The expression was measured using Image-J IHC profiler and summed as Optical density. The Optical density values were then subjected to statistical analysis. RESULTS: Results revealed a significant differential expression of Kaiso between the mucosal tissues taken from oral cancer patients and controls (p-value: < 0.0001), showing almost 50% down-regulation of Kaiso in all three tissue samples taken from oral cancer patients as compared to normal mucosa. CONCLUSION: Kaiso has a significant difference of expression in the mucosa of oral cancer patients as compared to the mucosa of normal patients, making it a probable contributor to disease pathogenesis and field cancerization.


Assuntos
Transformação Celular Neoplásica , Neoplasias Bucais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fatores de Transcrição , Caderinas/biossíntese , Caderinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Mucosa Bucal/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
16.
Sheng Wu Gong Cheng Xue Bao ; 38(9): 3228-3242, 2022 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-36151795

RESUMO

Recombinant collagen, as an alternative to natural collagen, has the potential to be widely used in biomaterials, biomedicine, etc. Diverse recombinant collagens and their variants can be industrially produced in a variety of expression systems, which lays a foundation for exploring and expanding the clinical application of recombinant collagens. We reviewed different expression systems for recombinant collagens, such as prokaryotic expression systems, yeast expression systems, as well as plant, insect, mammal, and human cell expression systems, and introduced the advantages, potential applications, and limitations of recombinant collagen. In particularly, we focused on the current progress in the recombinant collagen production, including recombinant expression system construction and hydroxylation strategies of recombinant collagen, and summarized the current biomedical applications of recombinant collagen.


Assuntos
Colágeno , Proteínas Recombinantes , Animais , Materiais Biocompatíveis , Colágeno/biossíntese , Humanos , Hidroxilação , Proteínas Recombinantes/biossíntese
17.
Biotechnol Lett ; 44(10): 1231-1242, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36074282

RESUMO

PURPOSE: Escherichia coli is an attractive and cost-effective cell factory for producing recombinant proteins such as single-chain variable fragments (scFvs). AntiEpEX-scFv is a small antibody fragment that has received considerable attention for its ability to target the epithelial cell adhesion molecule (EpCAM), a cancer-associated biomarker of solid tumors. Due to its metabolic burden, scFv recombinant expression causes a remarkable decrease in the maximum specific growth rate of the scFv-producing strain. In the present study, a genome-scale metabolic model (GEM)-guided engineering strategy is proposed to identify gene targets for improved antiEpEX-scFv production in E. coli. METHODS: In this study, a genome-scale metabolic model of E. coli (iJO1366) and a metabolic modeling tool (FVSEOF) were employed to find appropriate genes to be amplified in order to improve the strain for incresed production of antiEpEX-scFv. To validate the model predictions, one target gene was overexpressed in the parent strain Escherichia coli BW25113 (DE3). RESULTS: For improving scFv production, we applied the FVSEOF method to identify a number of potential genetic engineering targets. These targets were found to be localized in the glucose uptake system and pentose phosphate pathway. From the predicted targets, the glk gene encoding glucokinase was chosen to be overexpressed in the parent strain Escherichia coli BW25113 (DE3). By overexpressing glk, the growth capacity of the recombinant E. coli strain was recovered. Moreover, the engineered strain with glk overexpression successfully led to increased scFv production. CONCLUSION: The genome-scale metabolic modeling can be considered for the improvement of the production of other recombinant proteins.


Assuntos
Escherichia coli , Engenharia Metabólica , Anticorpos de Cadeia Única , Biomarcadores/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucoquinase , Glucose/metabolismo , Engenharia Metabólica/métodos , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/metabolismo
18.
Mol Biol (Mosk) ; 56(5): 795-807, 2022.
Artigo em Russo | MEDLINE | ID: mdl-36165018

RESUMO

Changes in metabolic pathways are often associated with the development of a wide range of pathologies. Increased glycolysis under conditions of sufficient tissue oxygen supply and its dissociation from the Krebs cycle, known as aerobic glycolysis or the Warburg effect, is a hallmark of many malignant neoplasms. Identification of specific metabolic shifts can characterize the metabolic programming of individual types of tumor cells, the stage of their transformation, and predict their metastatic potential. Viral infection can also alter the metabolism of cells to support the process of viral replication. Infection with human immunodeficiency virus type 1 (HIV-1) is associated with an increased incidence of various cancers, and for some viral proteins a direct oncogenic effect was demonstrated. In particular, we showed that the expression of HIV-1 reverse transcriptase (RT) in 4T1 breast adenocarcinoma cells increases the tumorigenic and metastatic potential of cells in vitro and in vivo by a mechanism associated with the ability of RT to induce reactive oxygen species in cells (ROS). The aim of this work was to study the molecular mechanism of this process, namely the effect of HIV-1 RT on the key metabolic pathways associated with tumor progression: glycolysis and mitochondrial respiration. Expression of HIV-1 RT had no effect on the glycolysis process. At the same time, it led to an increase in mitochondrial respiration and the level of ATP synthesis in the cell, while not affecting the availability of the substrates, carbon donors for the Krebs cycle, which excludes the effect of RT on the metabolic enzymes of cells. Increased mitochondrial respiration was associated with restoration of the mitochondrial network despite the RT-induced reduction in mitochondrial mass. Increased mitochondrial respiration may increase cell motility, which explains their increased tumorigenicity and metastatic potential. These data are important for understanding the pathogenesis of HIV-1 infection, including the stimulation of the formation and spread of HIV-1 associated malignancies.


Assuntos
Neoplasias da Mama , Carcinogênese , Transcriptase Reversa do HIV , HIV-1 , Mitocôndrias , Trifosfato de Adenosina/biossíntese , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/virologia , Carbono/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Respiração Celular , Ciclo do Ácido Cítrico , Feminino , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/metabolismo , Camundongos , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Nature ; 610(7931): 394-401, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36171293

RESUMO

Filoviruses, including Ebola virus, pose an increasing threat to the public health. Although two therapeutic monoclonal antibodies have been approved to treat the Ebola virus disease1,2, there are no approved broadly reactive drugs to control diverse filovirus infection. Filovirus has a large polymerase (L) protein and the cofactor viral protein 35 (VP35), which constitute the basic functional unit responsible for virus genome RNA synthesis3. Owing to its conservation, the L-VP35 polymerase complex is a promising target for broadly reactive antiviral drugs. Here we determined the structure of Ebola virus L protein in complex with tetrameric VP35 using cryo-electron microscopy (state 1). Structural analysis revealed that Ebola virus L possesses a filovirus-specific insertion element that is essential for RNA synthesis, and that VP35 interacts extensively with the N-terminal region of L by three protomers of the VP35 tetramer. Notably, we captured the complex structure in a second conformation with the unambiguous priming loop and supporting helix away from polymerase active site (state 2). Moreover, we demonstrated that the century-old drug suramin could inhibit the activity of the Ebola virus polymerase in an enzymatic assay. The structure of the L-VP35-suramin complex reveals that suramin can bind at the highly conserved NTP entry channel to prevent substrates from entering the active site. These findings reveal the mechanism of Ebola virus replication and may guide the development of more powerful anti-filovirus drugs.


Assuntos
Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA , Ebolavirus , Proteínas Virais Reguladoras e Acessórias , Antivirais/farmacologia , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Ebolavirus/enzimologia , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Viral/biossíntese , Suramina/química , Suramina/metabolismo , Suramina/farmacologia , Suramina/uso terapêutico , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Virais Reguladoras e Acessórias/ultraestrutura , Replicação Viral
20.
Nature ; 610(7932): 555-561, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36171294

RESUMO

CD4+ T cell differentiation requires metabolic reprogramming to fulfil the bioenergetic demands of proliferation and effector function, and enforce specific transcriptional programmes1-3. Mitochondrial membrane dynamics sustains mitochondrial processes4, including respiration and tricarboxylic acid (TCA) cycle metabolism5, but whether mitochondrial membrane remodelling orchestrates CD4+ T cell differentiation remains unclear. Here we show that unlike other CD4+ T cell subsets, T helper 17 (TH17) cells have fused mitochondria with tight cristae. T cell-specific deletion of optic atrophy 1 (OPA1), which regulates inner mitochondrial membrane fusion and cristae morphology6, revealed that TH17 cells require OPA1 for its control of the TCA cycle, rather than respiration. OPA1 deletion amplifies glutamine oxidation, leading to impaired NADH/NAD+ balance and accumulation of TCA cycle metabolites and 2-hydroxyglutarate-a metabolite that influences the epigenetic landscape5,7. Our multi-omics approach revealed that the serine/threonine kinase liver-associated kinase B1 (LKB1) couples mitochondrial function to cytokine expression in TH17 cells by regulating TCA cycle metabolism and transcriptional remodelling. Mitochondrial membrane disruption activates LKB1, which restrains IL-17 expression. LKB1 deletion restores IL-17 expression in TH17 cells with disrupted mitochondrial membranes, rectifying aberrant TCA cycle glutamine flux, balancing NADH/NAD+ and preventing 2-hydroxyglutarate production from the promiscuous activity of the serine biosynthesis enzyme phosphoglycerate dehydrogenase (PHGDH). These findings identify OPA1 as a major determinant of TH17 cell function, and uncover LKB1 as a sensor linking mitochondrial cues to effector programmes in TH17 cells.


Assuntos
Proteínas Quinases Ativadas por AMP , Mitocôndrias , Células Th17 , Glutamina/metabolismo , Interleucina-17/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Fosfoglicerato Desidrogenase/metabolismo , Serina/biossíntese , Serina/metabolismo , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Ciclo do Ácido Cítrico , GTP Fosfo-Hidrolases/deficiência , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo
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