scholarly journals Biotechnological Applications of Protein Splicing

2019 ◽  
Vol 20 (5) ◽  
pp. 408-424 ◽  
Author(s):  
Corina Sarmiento ◽  
Julio A. Camarero
Keyword(s):  
Protein Engineering ◽  
Chemical Biology ◽  
Protein Splicing ◽  
Biotechnological Applications ◽  
General Introduction ◽  
Recombinant Polypeptide ◽  
Affinity Tags ◽  
Novel Approaches

Protein splicing domains, also called inteins, have become a powerful biotechnological tool for applications involving molecular biology and protein engineering. Early applications of inteins focused on self-cleaving affinity tags, generation of recombinant polypeptide α-thioesters for the production of semisynthetic proteins and backbone cyclized polypeptides. The discovery of naturallyoccurring split-inteins has allowed the development of novel approaches for the selective modification of proteins both in vitro and in vivo. This review gives a general introduction to protein splicing with a focus on their role in expanding the applications of intein-based technologies in protein engineering and chemical biology.

scholarly journals Inhibiting TG2 sensitizes lung cancer to radiotherapy through interfering TOPOIIα-mediated DNA repair

2019 ◽  
Author(s):  
Xiao Lei ◽  
Zhe Liu ◽  
Kun Cao ◽  
Yuanyuan Chen ◽  
Jianming Cai ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Squamous Cell Carcinoma ◽  
Dna Repair ◽  
Transglutaminase 2 ◽  
Small Cell ◽  
Small Cell Lung ◽  
Poor Survival ◽  
Novel Approaches

AbstractRadiotherapy is an indispensable strategy for lung cancer, however, treatment failure or reoccurrence is often found in patients due to the developing radioresistance. Novel approaches are required for radiosensitizing to improve the therapeutic efficacy. In present study, we found that transglutaminase 2 (TG2) confers radioresistance in non-small cell lung cancer (NSCLC) cells through regulating TOPOIIα and promoting DNA repair. Our data showed that TG2 inhibitor or knockdown increased NSCLC radiosensitivity in vivo and in vitro. We found that TG2 translocated into nucleus and located to DSB sites, surprisingly, knockdown TG2 or glucosamine inhibited the phosphorylation of ATM, ATR and DNA-Pkcs. Through IP-MS assay and functional experiments, we identified that TOPOIIα as an downstream factor of TG2. Moreover, we found that TGase domain account for the interaction with TOPOIIα. Finally, we found that TG2 expression was correlated with poor survival in lung adenocarcinoma instead of squamous cell carcinoma. In conclusion, we demonstrated that inhibiting TG2 sensitize NSCLC to IR through interfere TOPOIIα mediated DNA repair, suggesting TG2 as a potential radiosensitizing target in NSCLC.

Recombinant Polypeptides Derived from the Fibrin Binding Domain of Fibronectin Are Potential Agents for the Imaging of Blood Clots

1997 ◽  
Vol 77 (04) ◽  
pp. 796-803 ◽  
Author(s):  
N Ezov ◽  
A Nimrod ◽  
B Parizada ◽  
M M Werber ◽  
A Goldlust ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Recombinant Polypeptide ◽  
Covalent Linkage ◽  
Steel Coil ◽  
Amino Terminal ◽  
Imaging Tool ◽  
Recombinant Polypeptides ◽  
Amino Terminal Domain

SummaryThrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa (“2 fingers”), 18.5 kDa (“3 fingers”) and 31 kDa FBD (“5 fingers”), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C- putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111ln]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [luIn]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

Chemical biology of in vivo and in vitro wounds

1968 ◽  
Vol 17 ◽  
pp. 19-26 ◽  
Author(s):  
J.C. Houck ◽  
J. Johnston ◽  
R.A. Jacob
Keyword(s):  
Chemical Biology

scholarly journals Protein engineering by highly parallel screening of computationally designed variants

2016 ◽  
Vol 2 (7) ◽  
pp. e1600692 ◽  
Author(s):  
Mark G. F. Sun ◽  
Moon-Hyeong Seo ◽  
Satra Nim ◽  
Carles Corbi-Verge ◽  
Philip M. Kim
Keyword(s):  
Protein Engineering ◽  
Protein Design ◽  
Tight Binding ◽  
Integrated Approach ◽  
Selection Strategies ◽  
Selection Systems ◽  
Protein Properties ◽  
Protein Binders

Current combinatorial selection strategies for protein engineering have been successful at generating binders against a range of targets; however, the combinatorial nature of the libraries and their vast undersampling of sequence space inherently limit these methods due to the difficulty in finely controlling protein properties of the engineered region. Meanwhile, great advances in computational protein design that can address these issues have largely been underutilized. We describe an integrated approach that computationally designs thousands of individual protein binders for high-throughput synthesis and selection to engineer high-affinity binders. We show that a computationally designed library enriches for tight-binding variants by many orders of magnitude as compared to conventional randomization strategies. We thus demonstrate the feasibility of our approach in a proof-of-concept study and successfully obtain low-nanomolar binders using in vitro and in vivo selection systems.

scholarly journals Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact

2021 ◽  
Author(s):  
Juha Rouvinen ◽  
Martina Andberg ◽  
Johan Pääkkönen ◽  
Nina Hakulinen ◽  
Anu Koivula
Keyword(s):  
Protein Engineering ◽  
Catalytic Mechanism ◽  
Cascade Reactions ◽  
Single Step ◽  
Enzyme Engineering ◽  
Enzymatic Reactions ◽  
Tim Barrel ◽  
The Stability

Abstract Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C–C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning–guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. Key points • DERA aldolases are versatile biocatalysts able to make new C–C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts. Graphical abstract

scholarly journals Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

2015 ◽  
Vol 112 (34) ◽  
pp. E4651-E4660 ◽  
Author(s):  
Miriam M. Cortese-Krott ◽  
Gunter G. C. Kuhnle ◽  
Alex Dyson ◽  
Bernadette O. Fernandez ◽  
Marian Grman ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Chemical Reactions ◽  
Chemical Biology ◽  
Arterial Compliance ◽  
Cardiac Contractility ◽  
Reaction Products ◽  
Hybrid Species ◽  
Redox Switching

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO−), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO− is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO− synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

scholarly journals Human ribosomal G-quadruplexes regulate heme bioavailability

2020 ◽  
Vol 295 (44) ◽  
pp. 14855-14865 ◽  
Author(s):  
Santi Mestre-Fos ◽  
Chieri Ito ◽  
Courtney M. Moore ◽  
Amit R. Reddi ◽  
Loren Dean Williams
Keyword(s):  
Chemical Biology ◽  
Human Cells ◽  
Affinity Reagents ◽  
In Cells

The in vitro formation of stable G-quadruplexes (G4s) in human rRNA was recently reported. However, their formation in cells and their cellular roles were not resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme-affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest that ribosomes play a role in regulating heme homeostasis.

scholarly journals O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

2020 ◽  
Vol 21 (8) ◽  
pp. 2878 ◽  
Author(s):  
Rosanna Mattossovich ◽  
Rosa Merlo ◽  
Riccardo Miggiano ◽  
Anna Valenti ◽  
Giuseppe Perugino
Keyword(s):  
Alkylating Agents ◽  
Basic Research ◽  
Living Cells ◽  
Alkylation Reaction ◽  
Biotechnological Applications ◽  
Human Homologue ◽  
Modern Biotechnology ◽  
Protein Tags

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

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